Expression of BMPRIA on human thymic NK cell precursors: role of BMP signaling in intrathymic NK cell development.

The bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34(+)CD1a(-)BMPRIA(+) precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34(+) cells mostly differentiate into functional CD56(+) natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15-responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

Disparate folding and stability of the ankylosing spondylitis-associated HLA-B*1403 and B*2705 proteins.

OBJECTIVE: To investigate the folding, assembly, maturation, and stability of HLA-B*1402 and B*1403, which differ by 1 amino acid change and are differentially associated with ankylosing spondylitis (AS), and to compare these features with those of B*2705. METHODS: Stable transfectants expressing B*1402, B*1403, and B*2705 were used. Folding rates were estimated from the ratio of unfolded heavy chains to folded heavy chains that had been immunoprecipitated with specific antibodies in pulse-chase experiments. Heavy chain misfolding was measured as the half-life of endoglycosidase H (Endo H)-sensitive beta2-microglobulin-free heavy chains. Maturation/export rates were measured by acquisition of Endo H resistance. Association with calnexin or tapasin was analyzed by coprecipitation with chaperone-specific antibodies, and surface expression was estimated by flow cytometry. Thermostability of HLA-peptide complexes was assessed by immunoprecipitation after incubation at various temperatures. Heavy chain expression was quantified by Western blotting. RESULTS: The folding rates of B*1402 and B*1403 were similar, and both were faster and more efficient than B*2705, but some unfolded heavy chains from both B14 subtypes remained in the endoplasmic reticulum (ER) with a long half-life. The export rates of B*1402 and B*1403 were slow, and the heterodimers partially dissociated after exiting the ER, as revealed by significant amounts of Endo H-resistant and surface-expressed free heavy chains. Both interaction with tapasin and thermostability were higher for B*2705 than for B*1402 and higher for B*1402 than for B*1403, suggesting that the repertoires of the B*1402-bound peptide and especially the B*1403-bound peptide were less optimized than that of B*2705. CONCLUSION: Our results indicate that the folding, maturation, and stability of B*1403 differ more from B*2705 than from B*1402. Thus, these features cannot account for the fact that only the 2 former allotypes are associated with AS.

Similar cell surface expression of beta2-microglobulin-free heavy chains by HLA-B27 subtypes differentially associated with ankylosing spondylitis.

OBJECTIVE: To determine whether the cell surface features of HLA-B27 subtypes reported to be differentially associated with ankylosing spondylitis (AS) differ in a way that correlates with disease susceptibility. METHODS: Human cell transfectants expressing or lacking the transporter associated with antigen processing were used to determine the cell surface expression of B27 subtypes by flow cytometry with antibodies recognizing the B27 heterodimer or beta2-microglobulin (beta2m)-free heavy chains. RESULTS: In lymphoid cells with an intact peptide-loading complex, all B27 subtypes, irrespective of their association with disease, showed similar ratios of free heavy chain to heterodimer, suggesting similar surface stability. A substantial decrease in dissociated heavy chains, which never reached 100%, was observed upon addition of a B27 ligand, with no significant differences among subtypes. This is compatible with similar surface expression of irreversible beta2m-free heavy chain forms among subtypes differentially associated with disease. In cells lacking the transporter associated with antigen processing, both disease-associated and non-disease-associated subtypes expressed a population of heterodimers at 26 degrees C that was less stable than the population expressed at 37 degrees C. In the presence of exogenous peptide, the expression of heterodimers increased, without a concomitant decrease in beta2m-free heavy chains. This suggests that in these cells, and for all subtypes tested, most of the dissociated heavy chains at the cell surface are in irreversible forms. At 37 degrees C, the expression of beta2m-free B27 heavy chains was very low on T2 transfectant cells. CONCLUSION: HLA-B27 subtypes showing differential associations with AS are similar in their extent of beta2m dissociation and surface expression of free heavy chains.