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Single-Cell, Phosphoepitope-Specific Analysis Demonstrates Cell Type- and Pathway-Specific Dysregulation of Jak/STAT and MAPK Signaling Associated with In Vivo Human Immunodeficiency Virus Type 1 Infection

Andrew W. Lee,^1,2, Elizabeth R. Sharp,³ Alison O'Mahony,⁴ Michael G. Rosenberg,⁵ Dennis M. Israelski,^2,6 Garry P. Nolan,^1, and Douglas F. Nixon^3*,

Baxter Laboratory of Genetic Pharmacology, Department of Microbiology and Immunology,¹ Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California 94305,² Division of Experimental Medicine, UCSF Box 1234, San Francisco, California 94143-1234,³ Gladstone Institute of Virology and Immunology, University of California at San Francisco, 1650 Owens Street, San Francisco, CA 94158-2261,⁴ Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, New York 10461,⁵ San Mateo County San Francisco AIDS Research Center (PARC), San Mateo County General Hospital and Health Department, San Mateo, California 94403⁶

Received 19 July 2007/ Accepted 8 January 2008

Despite extensive evidence of cell signaling alterations induced by human immunodeficiency virus type 1 (HIV-1) in vitro, the relevance of these changes to the clinical and/or immunologic status of HIV-1-infected individuals is often unclear. As such, mapping the details of cell type-specific degradation of immune function as a consequence of changes to signaling network responses has not been readily accessible. We used a flow cytometric-based assay of signaling to determine Janus kinase/signal transducers and activators of transcription (Jak/STAT) signaling changes at the single-cell level within distinct cell subsets from the primary immune cells of HIV-1-infected donors. We identified a specific defect in granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven Stat5 phosphorylation in the monocytes of HIV-1+ donors. This inhibition was statistically significant in a cohort of treated and untreated individuals. Ex vivo Stat5 phosphorylation levels varied among HIV-1+ donors but did not correlate with CD4⁺ T-cell counts or HIV-1 plasma viral load. Low Stat5 activation occurred in HIV-1-infected donors despite normal GM-CSF receptor levels. Investigation of mitogen-activated protein kinase (MAPK) pathways, also stimulated by GM-CSF, led to the observation that lipopolysaccharide-stimulated extracellular signal-regulated kinase phosphorylation is enhanced in monocytes. Thus, we have identified a specific, imbalanced monocyte signaling profile, with inhibition of STAT and enhancement of MAPK signaling, associated with HIV-1 infection. This understanding of altered monocyte signaling responses that contribute to defective antigen presentation during HIV-1 infection could lead to immunotherapeutic approaches that compensate for the deficiency.

Published ahead of print on 23 January 2008.

Present address: Merck Research Laboratories, Infectious Diseases/Vaccines Clinical Research, 351 Sumneytown Pike, North Wales, PA 19454.

G.P.N. and D.F.N. contributed equally to this study.