Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation

Abstract

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.

Deregulated Mucosal Immune Surveillance through Gut-Associated Regulatory T Cells and PD-1+ T Cells in Human Colorectal Cancer

“…Human CD8 + T cells from normal mucosa and CD4 + CD25 + CD127 2 Tregs and CD4 + CD25 2 CD127 + conventional T cells (Tconvs) from adenocarcinoma were purified by FACS using FACSAria (BD Biosciences). A total of 5-8 3 10 4 CD8 + T cells were labeled with 7.5 mM CFSE for 10 min at 37˚C and cocultured with the same numbers of either Tconvs or Tregs for 5 d in RPMI 1640 supplemented with 10% FBS in the presence of anti-CD3/CD28 beads at equal numbers to CTLs (Invitrogen) (22)(23)(24)(25). On day 5 of coculture, total cell numbers were obtained, expansion of human T cells were observed by light microscopy using Zeiss Axiovert 200 (Carl Zeiss), and the dilution of CFSE in CD8 + T cells was evaluated by FACSCanto II (BD Biosciences).…”

A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

“…Ex vivo stimulation of T cells with HCMV antigens has shown promise as an alternative to traditional vaccination (14), but this technology is cumbersome to implement on a commercial scale. Moreover, the antiviral activity of T cells is inhibited by granulocyte colonystimulating factor (G-CSF) (15), which is commonly used to promote the engraftment of hematopoietic stem cells.…”

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood

“…Most protocols have used non-mobilized apheresis samples as starting material, but the manufacture of the therapeutic product from the same G-CSF mobilized sample used for PBSC collection offers great logistical and regulatory advantages that could avoid the need for successive blood donations and consequently widen the applicability of the adoptive immunotherapy. Recently, the feasibility of CMV-specific T cell generation from a G-CSF mobilized collection has been assessed, confirming the applicability of the procedure [ 6 – 8 , 18 ]. Despite these new findings, there is a concern about the effectiveness of the generated cell products due to the immunosuppressive effects associated with G-CSF administration [ 9 , 19 , 20 ].…”

“…We hypothesize that potential inadequacies in the in vitro proliferation protocol resulted in reduced proliferation of G-CSF mobilized CMV-CTLs and that different culture conditions may improve proliferation. Furthermore, in our previous studies where CMV-specific T cells were isolated through CD25, CD137, or CD154 activation marker expression and expanded in the same conditions described here [ 6 , 8 , 18 ], cells isolated from G-CSF mobilized PBMCs were able to proliferate as efficiently as non-mobilized CMV-specific T cells. In these cases, the cellular products were made of CD4+ and CD8+ cell subsets.…”

“…The majority of studies that generated virus-specific T cells have used non-mobilized peripheral blood mononuclear cells (PBMCs) isolated from an additional leukapheresis collection from the original allo-HSCT donor, different to that of the peripheral blood stem cell (PBSCs) collection performed after granulocyte-colony stimulating factor (G-CSF; Filgrastim) treatment. This additional leukapheresis harvest, apart from increasing the cost of the procedure and the discomfort caused to the donor, can be difficult to obtain in the unrelated donor setting where donor refusal or logistical and scheduling difficulties can prevent collection [ 6 – 8 ]. Manufacture of virus-specific T cells from the original G-CSF mobilized collection could potentially overcome the difficulties associated with procurement of a second apheresis.…”