Clinical-scale isolation of the total Aspergillus fumigatus-reactive T-helper cell repertoire for adoptive transfer

Cytotherapy. 2015 Oct;17(10):1396-405. doi: 10.1016/j.jcyt.2015.05.011. Epub 2015 Jul 15.


Background aims: Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective.

Methods: We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137.

Results: In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 × 10(9) peripheral blood mononuclear cells, we isolated 27 × 10(3)-318 × 10(3)Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens.

Discussion: Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-specific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 × 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific.

Keywords: GMP-compliant protocol; T-helper cells; aspergillosis; cell therapy; prospective isolation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Fungal / immunology
  • Aspergillosis / immunology
  • Aspergillosis / therapy*
  • Aspergillus fumigatus / immunology*
  • Candida albicans / immunology
  • Cell Separation / methods*
  • Cytokines / immunology
  • Hematopoietic Stem Cell Transplantation / adverse effects
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Leukocytes, Mononuclear / immunology
  • Lymphocyte Activation / immunology*
  • Lymphocyte Depletion / methods
  • T-Lymphocytes, Helper-Inducer / immunology
  • T-Lymphocytes, Helper-Inducer / transplantation*
  • Tumor Necrosis Factor Receptor Superfamily, Member 9 / metabolism


  • Antigens, Fungal
  • Cytokines
  • Tumor Necrosis Factor Receptor Superfamily, Member 9