Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.

In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation.

Pretransplant helper T-lymphocyte determination in bone marrow donors: acute graft-versus-host disease prediction and relation with long-term survival.

Helper T-lymphocyte precursor (HTLp) frequency from 19 allogeneic bone marrow donors was tested to detect weak antigenic differences with the recipient, and then compared to the outcome. HTLp frequency was estimated in limiting dilution cultures, and HLA-DR and CD 80 expression by stimulating cells was measured by flow cytometry. 12/19 patients experienced acute graft-versus-host disease (aGVHD) grade II-IV. A good correlation was found between high pretransplant HTLp frequency and grade II-IV aGVHD (median: 1/55848 PBMNC for II-IV GVHD versus 1/184346 for 0-I GVHD; P = 0.008). Sensitivity was 82%, specificity 63%, negative predictive value 71% and positive predictive value 75%. Long-term survivors also had a lower HTLp median frequency (1/143354) when compared with patients who died as a result of the transplant procedure (1/22100, P < 0.001). No correlation was found between HTLp frequency and HLA-DR or CD80 expression by patient's cells. We conclude that HTLp frequency estimation can predict, although poorly, acute GVHD risk and long-term survival.

Improved IL-2 detection for determination of helper T lymphocyte precursor frequency in limiting dilution assay.

In the context of allogeneic bone marrow transplantation, an accurate estimate of the risk of developing graft-versus-host disease (GVHD) is of major interest. The pre-transplant frequency of donor's helper T-lymphocyte precursors (HTLp) directed against host's antigens may be helpful in predicting this risk. This technique relies on an indirect measurement of interleukin-2 (IL-2) secreted by the HTLp, as assessed by the proliferation of an IL-2 dependent cell line. Many authors use the murine CTLL-2 cell line in this assay, but these cells do not respond to the presence of minute amounts of IL-2 in the culture medium, and thus do not discriminate between the absence or the presence of very low levels of IL-2. We therefore decided to compare CTLL-2 with another IL-2 dependent cell line, the murine A9.12 cell line. A comparison was made using serial dilutions of recombinant human IL-2, limiting dilutions of baby hamster kidney (BHK) cells transfected with human IL-2 gene and in the context of clinical tests performed for the detection of pre-transplant HTLp. Both the sensitivity and reliability of the tests were better using A9.12. We conclude that the A9.12 cell line might be a more suitable tool for pre-transplant HTLp determinations before allogeneic bone marrow transplantation or whenever low IL-2 levels are to be measured.