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ABSTRACT: The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have therefore generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 109 putative Tregs co-expressing CD25 and CD4 molecules (92%+/-5%) and FoxP3 (69%+/-19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80%+/-13% inhibition of CD8+ cell division) and in vivo (suppression or delay of GvHD in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.
Haematologica 12/2012; · 6.42 Impact Factor
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Serena K Perna,
Biagio De Angelis,
Daria Pagliara,
Sayyeda T Hasan,
Lan Zhang,
Aruna Mahendravada,
Helen E Heslop,
Malcolm K Brenner,
Cliona M Rooney,
Gianpietro Dotti,
Barbara Savoldo
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ABSTRACT: PURPOSE: Systemic administration of recombinant IL-2 is used to support the expansion and persistence of adoptively transferred antigen-specific cytotoxic T lymphocytes (CTLs) in cancer patients. However, IL-2 also expands regulatory T cells (Tregs) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein-Barr-Virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin Lymphoma (HL), and Tregs are frequently abundant in these patients. EXPERIMENTAL DESIGN: Tregs were isolated from the peripheral blood of healthy donors and HL patients or from HL tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. RESULTS: We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs, but instead preferentially supported the proliferation of CTLs and effector T cells as compared to Tregs. CONCLUSIONS: IL-15 selectively favours the survival, proliferation and effector function of antigen-specific CTLs in the presence of Tregs and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in cancer patients including those infused with EBV-CTLs for treatment of EBV-associated malignancies.
Clinical Cancer Research 11/2012; · 7.74 Impact Factor
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Ulrike Gerdemann,
Jacqueline M Keirnan,
Usha L Katari,
Ryu Yanagisawa,
Anne S Christin,
Leslie E Huye, Serena K Perna,
Sravya Ennamuri,
Stephen Gottschalk,
Malcolm K Brenner,
Helen E Heslop,
Cliona M Rooney,
Ann M Leen
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ABSTRACT: Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4(+) and CD8(+)) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.
Molecular Therapy 07/2012; 20(8):1622-32. · 6.87 Impact Factor
