Rapamycin and interleukin-10 treatment induces T regulatory type 1 cells that mediate antigen-specific transplantation tolerance.

Islet transplantation is a cure for type 1 diabetes, but its potential is limited by the need for constant immunosuppression. One solution to this problem is the induction of transplantation tolerance mediated by T regulatory cells. T regulatory type 1 (Tr1) cells are characterized by their production of high levels of interleukin (IL)-10, which is crucial for their differentiation and suppressive function. We investigated the effects of IL-10 administered in combination with rapamycin on the induction of Tr1 cells that could mediate a state of tolerance in diabetic mice after pancreatic islet transplantation. The efficacy of this treatment was compared with IL-10 alone and standard immunosuppression. Stable long-term tolerance that was not reversible by alloantigen rechallenge was achieved only in mice treated with rapamycin plus IL-10. Tr1 cells that produced high levels of IL-10 and suppressed T-cell proliferation were isolated from splenocytes of rapamycin plus IL-10-treated mice after treatment withdrawal. In rapamycin plus IL-10-treated mice, endogenous IL-10 mediated an active state of tolerance, as was observed when the blockade of IL-10 activity rapidly induced graft rejection >100 days after transplantation. CD4(+) T-cells from rapamycin plus IL-10-treated mice transferred antigen-specific tolerance in mice that received new transplants. Thus rapamycin plus IL-10 not only prevented allograft rejection but also induced Tr1 cells that mediated stable antigen-specific, long-term tolerance in vivo.

IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34+ cells while maintaining in vivo lymphoid potential.

To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.

Mobilized blood CD34+ cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma.

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.