T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.
In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.
The herpes simplex virus thymidine kinase ( HSV -tk ) gene conferring ganciclovir ( GCV ) -specific sensitivity to transduced cells might control Graft -versus -Leukemia ( GvL ) / Graft -versus -Host Disease ( GvHD ). Human T lymphocytes were engineered with an LSN -tk retroviral vector encoding tk and neomycin resistance ( NeoR ) genes. A total of 80Â10 6 tk + lymphocytes were injected intraperitoneally in NOD -SCID mice. Engraftment was evaluated by human CD45 + / CD3 + cytofluorimetric analysis and NeoRbased polymerase chain reaction ( PCR ) on peripheral blood, bone marrow, liver, thymus, and spleen on day + 5. After 14 days, GCV ( 10 mg / kg daily ) cytofluorimetric analysis and PCR were repeated ( day + 19 ). Immunohistological studies with anti -CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti -alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45 + / CD3 + cells were engrafted in all tissues on day + 5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes ''homed'' to the white pulp T -cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV ( day + 19 ), cytofluorimetry and immunohistology showed very few CD3 + cells. PCR identified the transgene in 22% tissue samples ( positive only in thymus and spleen ). GvHD did not occur in any animal. These data demonstrate elevated doses of human -transduced CD3 + cells engraft in NOD / SCID mice; after GCV, very few CD3 + cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day + 19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk -transduced T lymphocytes.
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