Successful isolation and expansion of CMV‐reactive T cells from G‐CSF mobilized donors that retain a strong cytotoxic effector function

Abstract

SummaryCytomegalovirus (CMV) infections post-haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor-derived CMV-specific T cells (CMV-T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV-T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte-colony stimulating factor (G-CSF) mobilized HSCT graft to manufacture CMV-T. We explored the T cell response to CMVpp65 peptide stimulation in G-CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV-T based on the activation markers CD154 and CD25. CD25 + enriched CMV-T from G-CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non-mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV-T enriched through CD154 were CD4+ and CD8 + , demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. These data provide the first known evidence that CMV-T can be effectively manufactured from G-CSF mobilized PBMC and that they share the same characteristics as CMV-T isolated in an identical manner from conventional non-mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation.

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

“…Generation of anti-viral T cells has been largely restricted to non-mobilized steady state leukapheresis or a single blood draw for manufacture. More recently, studies have investigated the use of G-CSF-mobilized PBMCs from the original aHSCT graft as a potential source for CMV-T manufacture [31]; [32]. Here, we expand on these studies to investigate the activation marker CD25 as a possible target and investigate the nature of CD25+ enriched cells following CMV peptide stimulation with regard to Treg characterisation, due to their increased number following G-CSF-mobilization [36].…”

“…These results alleviate some of the major concerns surrounding the feasibility of using G-CSF-mobilized lymphocytes as a starting material for the manufacture of anti-viral immunotherapies. We have published our previous findings that demonstrated the feasibility of employing the use of G-CSF-mobilized PBMCs as an effective strategy for manufacture of CMV-T with the retention of functional CMV-specific cytotoxicity comparable with non-mobilized PBMCs, using CD154 based selection [31]. But the translation of this particular approach is currently restricted by the non-availability of a GMP-compliant anti-CD154 antibody.…”

Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy

“…While G-CSF can increase the number of circulating lymphocytes (40–42), the observation that G-CSF inhibits the production of Th1-type cytokines raised concerns on the use of mobilized T cells with respect to immune reconstitution (43–47). Nevertheless, T cells from mobilized donors proved suitable for specific selection using either multimers (48, 49) or activation markers for enrichment and expansion (50, 51), with effectiveness comparable to that of T cells from non-mobilized samples.…”

Humanes Cytomegalievirus (HCMV)

“…B. bei haploidenter Transplantation) beinhalten. Einige Studien konnten die Effizienz in der Therapie und die Vermeidung des Auftretens von HCMV-Erkrankungen nachweisen [91,92,96,100,[104][105][106][107]. Die Gabe von virusspezifischen Spender-T-Lymphozyten einige Wochen nach Transplantation senkt zusätzlich das Risiko des Auftretens einer GVHD, ohne dass die Ursachen dafür bisher bekannt sind [58,108].…”

“…Unklar ist derzeit, ob antigenspezifische CD4-oder CD8-positive T-Zellen einzeln oder gemeinsam appliziert werden sollten [105]. Erwähnenswert ist der Ansatz, die Expansion aus einem kleinen Aliquot des Stammzelltransplantats vorzunehmen und damit die logistisch oft problematische Wiedereinbestellung des Spenders zur Gewinnung virusspezifischer T-Zellen zu vermeiden [106,107]. Die Fortschritte auf diesem Gebiet sind vielversprechend [109].…”