The Axl/Gas6 pathway is required for optimal cytokine signaling during human natural killer cell development.

Interleukin-15 (IL-15) is essential for natural killer (NK) cell differentiation. In this study, we assessed whether the receptor tyrosine kinase Axl and its ligand, Gas6, are involved in IL-15-mediated human NK differentiation from CD34(+) hematopoietic progenitor cells (HPCs). Blocking the Axl-Gas6 interaction with a soluble Axl fusion protein (Axl-Fc) or the vitamin K inhibitor warfarin significantly diminished the absolute number and percentage of CD3(-)CD56(+) NK cells derived from human CD34(+) HPCs cultured in the presence of IL-15, probably resulting in part from reduced phosphorylation of STAT5. In addition, CD3(-)CD56(+) NK cells derived from culture of CD34(+) HPCs with IL-15 and Axl-Fc had a significantly diminished capacity to express interferon-gamma or its master regulator, T-BET. Culture of CD34(+) HPCs in the presence of c-Kit ligand and Axl-Fc resulted in a significant decrease in the frequency of NK precursor cells responding to IL-15, probably the result of reduced c-Kit phosphorylation. Collectively, our data suggest that the Axl/Gas6 pathway contributes to normal human NK-cell development, at least in part via its regulatory effects on both the IL-15 and c-Kit signaling pathways in CD34(+) HPCs, and to functional NK-cell maturation via an effect on the master regulatory transcription factor T-BET.

Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells.

Natural killer (NK) cells contribute to host immunity, including tumor surveillance, through the production of interferon gamma (IFN-gamma). Although there is some knowledge about molecular mechanisms that induce IFN-gamma in NK cells, considerably less is known about the mechanisms that reduce its expression. Here, we investigate the role of the Hlx transcription factor in IFN-gamma production by NK cells. Hlx expression is induced in monokine-activated NK cells, but with delayed kinetics compared to IFN-gamma. Ectopic Hlx expression decreases IFN-gamma synthesis in primary human NK cells and IFN-gamma promoter activity in an NK-like cell line. Hlx protein levels inversely correlate with those of STAT4, a requisite factor for optimal IFN-gamma transcription. Mechanistically, we provide evidence indicating that Hlx overexpression accelerates dephosphorylation and proteasome-dependent degradation of the active Y693-phosphorylated form of STAT4. Thus, Hlx expression in activated NK cells temporally controls and limits the monokine-induced production of IFN-gamma, in part through the targeted depletion of STAT4.

HLA antibodies and soluble CD30 are associated with poor renal graft outcome: updated results of a single-center cross-sectional study.

In a previous study, we have shown that HLA class II antibodies and a high soluble CD30 (sCD30) measured at least 1 year post-transplant predict subsequent graft failure. We have now updated the results of this same cohort of 208 patients 15 months later. HLA-specific antibodies (class I and class II) were detected by ELISA LAT-M and Luminex LabScreen assays. Data on graft outcome was collected with a median follow-up of 4.7 years. By Kaplan-Meier analysis, class II antibody was again associated with a poorer outcome, with an estimated 6-year graft survival of 67% and 71% when detected by ELISA and Luminex, respectively, compared with 92% for those without class II antibody (p < or = 0.0001). A soluble CD30 level of > or = 100 U/ml was also associated with a poorer estimated 6-year graft survival (p = 0.02). HLA antibodies and high sCD30 (> or = 100 U/ml) had an additive effect such that those with both high sCD30 and class II antibodies had a hazard ratio for subsequent graft failure of 18.1 (p = 0.0008) and 8.6 (p = 0.007) when detected by ELISA and Luminex, respectively. These data show that detection of HLA class II antibodies and serum sCD30 measured at least 1 year post-transplant continues to predict a subsequent outcome up to 6 years after the initial measurement; they also show that such measures provide important information that may allow for modification of ongoing therapy.

A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells.

In humans, T cells differentiate in thymus and B cells develop in bone marrow (BM), but the natural killer (NK) precursor cell(s) and site(s) of NK development are unclear. The CD56bright NK subset predominates in lymph nodes (LN) and produces abundant cytokines compared to the cytolytic CD56dim NK cell that predominates in blood. Here, we identify a novel CD34dimCD45RA(+) hematopoietic precursor cell (HPC) that is integrin alpha4beta7bright. CD34dimCD45RA(+)beta7bright HPCs constitute <1% of BM CD34(+) HPCs and approximately 6% of blood CD34(+) HPCs, but >95% of LN CD34(+) HPCs. They reside in the parafollicular T cell regions of LN with CD56bright NK cells, and when stimulated by IL-15, IL-2, or activated LN T cells, they become CD56bright NK cells. The data identify a new NK precursor and support a model of human NK development in which BM-derived CD34dimCD45RA(+)beta7bright HPCs reside in LN where endogenous cytokines drive their differentiation to CD56bright NK cells in vivo.