A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase

A sensitized genetic system for the analysis of murine B lymphocyte signal transduction pathways dependent on Bruton's tyrosine kinase

^aDepartment of Microbiology, Immunology, and Molecular Genetics, and ^cHoward Hughes Medical Institute, University of California, Los Angeles, CA 90095; ^dDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; ^eDivision of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02215; ^fTerry Fox Laboratories, British Columbia Cancer Agency, Vancouver, BC, V57 IL3, Canada; ^gDepartment of Laboratory Medicine, University of California, San Francisco, CA 94143; ^hDivision of Biology, California Institute of Technology, Pasadena, CA 91125; ⁱMax Planck Institute for Immunobiology, Stöberweg 51, 79108, Freiburg Germany; ^jInstitute for Genetics, University of Cologne, Weyertal 121, 50931, Cologne, Germany; ^kDepartment of Immunology, Duke University Medical Center, Durham, NC 27710; and ^lDepartment of Molecular and Medical Pharmacology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095

Contributed by Owen N. Witte

Abstract

Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk^lo) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk^−/− mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk^lo phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk^lo phenotype in vitro but not in vivo, whereas CD19 and the p85α form of phosphoinositide 3-kinase behaved as Btk^lo enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85α haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C β, Fyn, CD22, Gαq, or Gα11 had no detectable effect on the function of Btk^lo B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.

Footnotes

↵ b Present address: Harold C. Simmons Arthritis Research Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884.
↵ m To whom reprint requests should be addressed at: Howard Hughes Medical Institute, University of California, Los Angeles, 5-720 MacDonald Research Laboratories, 675 Charles E. Young Drive South, Los Angeles, CA 90095. E-mail: owenw{at}microbio.ucla.edu.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.110146697.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.110146697
Abbreviations:

Btk,

Bruton's tyrosine kinase;

Btklo,

Btk transgenic mice expressing 25% of endogenous Btk levels;

TI-II,

type II T cell independent antigen;

xid,

X-linked immunodeficiency;

BCR,

B cell antigen receptor;

TNP,

2,4,6-trinitrophenyl;

PI3K,

phosphoinositide 3-kinase;

PIP3,

phosphatidylinositol 3,4,5-triphosphate;

FITC,

fluorescein isothiocyanate;

PE,

phycoerythrin;

SHIP,

SH2-containing inositol phosphatase;

PTEN,

phosphatase and tensin homolog;

PKCβ,

protein kinase Cβ