Ex-vivo treatment of peripheral plasmacytoid dendritic cells with IFN-lambda

#The present invention generally relates to a method of ex vivo treatment of an immune cell by interferon lambda (IFN-λ). Specifically, the present invention provides a method of isolating peripheral blood leukocytes from a human subject, and further isolating plasmacytoid dendritic cells (pDC), and exposing the isolated pDC to IFN-λ at a concentration sufficient to reduce the pDC-support T-cell production of specific cytokines (i.e., IFN-γ, IL-13, IL-10 and IL-17), and administering the IFN-λ treated pDC into the human subject. The ex vivo treatment of pDC by IFN-λ reduces Th2 and Th17 responses, probably via its hitherto unexpected ability to modulate co-stimulatory molecules as well as homing molecules present in pDC. The ex vivo treatment may be combined with an administration of IFN-λ. The present invention is applicable to patients having Th2- and Th17-associated diseases such as asthma, inflammatory bowel diseases including Crohn's disease.

#Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disorder that affects >1.4 million people in the United States. Symptoms of IBD include abdominal pain, weight loss, fever, and diarrhea, resulting a significant mortality and morbidity. IBD includes Crohn's disease and ulcerative colitis. There is presently no cure for IBD. Treatment involves administration of sulfa drugs, corticosteroids, immuno-suppressives and anti-tumor necrosis factor agents. Patients often develop non-responsiveness to these drug treatments, and over time surgery represents the only option. Inflamed sections of the gut (e.g., colon) are resected, and connected to adjacent sections (e.g., ileoanal anastomosis). However, surgery does not guarantee successful treatment.

#The underlying immunologic basis for IBD is complex and is not completely understood. IBD is a persistent inflammation in the gut that is driven by immune cells (e.g., T-cells), which are capable of sustaining a chronic production of inflammatory cytokines. In Crohn's disease, one of the major T-cell subtypes that contributes to disease is the Th17 cell, so named because of the production of the IL-17 cytokine family (e.g., IL-17A, IL-17F, IL-21, IL-22). The IL-17 cytokines may act in concert with other cytokines (e.g., IL-1, IL-13, IL-10, etc) to yield persistent and invasive inflammatory lesions. The chronic inflammation that drives IBD is significantly different from other types of inflammation (e.g., TNF and IL-1), including that which occurs in Irritable Bowel Syndrome (IBS), a less serious intestinal inflammatory disorder.

#FIG. 1 shows that IFN-λ lowers the IL-4 and IL-13 mRNA levels. Panel A (IL-4 mRNA levels at 24 hours); Panel B (IL-5 mRNA levels at 24 hours); Panel C (IL-13 mRNA levels at 24 hours); Panel D (IL-4 mRNA levels at 6 days); Panel E (IL-5 mRNA levels at 6 days); Panel F (IL-13 mRNA levels at 6 days).

#FIG. 2 shows that IFN-λ, lowers secreted levels of IL-4, IL-5 and IL-13. Panel A (IL-4 levels at 24 hours); Panel B (IL-5 levels at 6 days); Panel C (IL-13 levels at 6 days).

#FIG. 3A shows that IFN-λ reduced the numbers of cytokine positive CD4 T-cells. Panel A, FACS analysis of CD4+ and CD3+ cells; Panel B, FACS analysis of IL-4+ and IL-5+ cells (6 hours without IFN-λ1 added); Panel C, FACS analysis of IL-13+ and IFN-γ+ cells (6 days without IFN-λ added); Panel D, FACS analysis of IL-4+- and IL-5+ cells (6 hours with IFN-λ, added); Panel E, FACS analysis of IL-13+ and IFN-γ+ cells (6 days with IFN-λ, added).

#All patents, patent applications, published literature and citations to the NCBI database Accession Numbers cited herein are incorporated by reference in their entirety including all amino acid sequence data therein.

#Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1998; Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED., Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1989; Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton, 1995; McPherson, Ed.

#As used herein, the term “interferon lambda” (i.e., IFN-λ) is used interchangeably to describe a family of proteins that include IFN-λ1 (IL-29); IFN-λ2 (IL-28A); IFN-λ3 (IL-28B). These proteins are termed “Type III” interferons (Kotenko et al., 2003, Nat. Immunol., 4, 69-77). For purposes of this application, IFN-λ is intended to encompass all members of the IFN-λ family including, for example, IFN-λ1, IFN-λ2, IFN-λ3, and the variants thereof insofar as these molecules function equivalently in the methods of the invention in inhibiting cytokine production of IL-4, IL-5 and IL-13 from immune cells. Signaling is mediated through a heterodimeric receptor complex composed of the signaling subunit, IL-28Rα and the non-signaling chain, IL-10Rβ; all three ligands signal through this receptor.