Michael G. Kharas

Sjögren's syndrome-like disease in mice with T cells lacking class 1A phosphoinositide-3-kinase

Sjögrens syndrome (SS) is an autoimmune disease that is characterized by infiltration of exocrine tissues, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Here, we show that mice with T cell-specific loss of class IA phosphoinositide 3-kinase function develop organ-specific autoimmunity that resembles the human disease SS. Most mutant mice aged 3–8 months develop corneal opacity and eye lesions due to irritation and constant scratching. These mice display cardinal signs of primary SS such as marked lymphocytic infiltration of the lacrimal glands, antinuclear antibodies in the serum, and elevated titer of anti-SS-A antibody, in the absence of kidney pathology. Immunofluorescence studies show the presence of numerous CD4+ T cells with a smaller number of CD8+ T cells and B cells in the lacrimal glands. CD4+ T cells from these mice exhibit aberrant differentiation in vitro. These results indicate that aberrant T cells with impaired class IA phosphoinositide 3-kinase signaling can lead to organ-specific autoimmunity. In addition, the mouse model described here represents a tool to study the pathogenesis and treatment of SS.

KLF4 is a FOXO target gene that suppresses B cell proliferation

Lymphocytes circulate in a quiescent (G(0)) state until they encounter specific antigens. In T cells, quiescence is programmed by transcription factors of the forkhead box O (FOXO) and Krüppel-like factor (KLF) families. However, the transcription factors that regulate B cell quiescence are not known. KLF4 is a candidate tumor suppressor gene in B lymphocytes, and thus a likely candidate for regulating B cell homeostasis. Here, we show that RNA and protein expression of murine KLF4 decreases following B cell activation. Forced expression of KLF4 in proliferating B cell blasts causes a G(1) cell cycle arrest. This effect requires the DNA binding and transactivation domains of KLF4 and correlates with changes in the expression of known KLF target genes. We present evidence that Klf4 is a target gene for FOXO transcription factors, which also suppress B cell proliferation. To determine the effect of KLF4 loss-of-function, we generated mice with B cell-specific deletion of the Klf4 gene. These mice exhibited normal B cell development and function with no evidence of a lowered activation threshold. Collectively, our findings indicate that KLF4 has growth-suppressive properties in B cells but might be redundant with other members of the KLF family in maintaining B cell quiescence.

S6 kinase 2 potentiates interleukin-3-driven cell proliferation

Interleukin‐3 (IL‐3) mediates hematopoietic cell survival and proliferation via several signaling pathways such as the Janus kinase/signal transducer and activator of transcription pathway, mitogen‐activated protein kinase (MAPK) pathway, and phosphoinositide‐3 kinase (PI‐3K) pathway. Mammalian target of rapamycin (mTOR) is one of the downstream targets of the PI‐3K pathway, and it plays an important role in hematopoiesis and immune cell function. To better elucidate how mTOR mediates proliferation signals from IL‐3, we assessed the role of S6 kinase 2 (S6K2), one of the downstream targets of mTOR, in IL‐3 signaling. We show that S6K2 is activated by IL‐3 in the IL‐3‐dependent Ba/F3 cell line and that this is mediated by mTOR and its upstream activator PI‐3K but not by the MAPK kinase/extracellular signal‐regulated kinase pathway. S6K2 is also activated in primary mouse bone marrow‐derived mast cells upon IL‐3 stimulation. Expression of a rapamycin‐resistant form of S6K2, T388E, in Ba/F3 cells provides a proliferation advantage in the absence or presence of rapamycin, indicating that S6K2 can potentiate IL‐3‐mediated mitogenic signals. In cells expressing T388E, rapamycin still reduces proliferation at all doses of rapamycin, showing that mTOR targets other than S6K2 play an important role in IL‐3‐dependent proliferation. Cell‐cycle analysis shows that T388E‐expressing Ba/F3 cells enter S phase earlier than the control cells, indicating that the proliferation advantage may be mediated by a shortened G1 phase. This is the first indication that S6K2 plays a role in IL‐3‐dependent cell proliferation.

Measuring phosphorylated Akt and other phosphoinositide 3-kinase-regulated phosphoproteins in primary lymphocytes.

Phosphoinositide 3-kinase (PI3K) is a lipid kinase whose activation is crucial for many biological functions in multiple cell types. One research area of particular interest for basic biologists and drug developers is PI3K signaling in lymphocytes. Inhibitor studies and PI3K mutants have demonstrated that PI3K is required for development, activation, proliferation, differentiation, and survival of B lymphocytes, as well as optimal activation and proliferation of T lymphocytes. As the actual products of PI3K can be difficult to measure, the field has often adopted the practice of examining the activation of downstream effectors of PI3K, with the most common readout being phosphorylation of Akt. This chapter discusses key pathways influenced by PI3K signaling and the advantages and caveats of using activation of these pathways as indicators of PI3K activity. In addition, we provide traditional immunoblotting methods of assaying PI3K-dependent pathway activation, as well as more recent flow cytometry-based approaches (termed phosflow). Although we describe assays optimized for B lymphocytes, these methods are easily adapted to T lymphocytes and other leukocyte cell types.