Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-ß, granzyme B). TIM3+ cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Evaluation of an automated closed fluid management device for processing expanded cytokine-induced killer cells to use in immunotherapy programs for cancer.

BACKGROUND: Cytokine-induced killer (CIK) cells are a heterogeneous population of immune cells derived from peripheral blood lymphocytes with a high proliferative potential ex vivo. This study shows a rapid and reproducible protocol for adoptive immunotherapy with CIK cells in patients with hematologic malignancies. For this purpose a new automatic cell processing device (CytoMate, Baxter Oncology) was tested to improve extensive manipulations of these cells. STUDY DESIGN AND METHODS: Twenty CIK expansions obtained from healthy donors and patients with hematologic malignancies were washed and refilled with fresh medium during culture with the CytoMate. Recovery, viability, and cytotoxic activity were evaluated. Six cryopreserved CIK procedures were thawed and processed for washing out dimethyl sulfoxide automatically. Recovery of cells, viability, and early apoptosis were measured immediately after washing, and cytotoxic activity against target cell lines K562 and Daudi was tested after short culture. RESULTS: Prewash volume of CIK cultures was 3600 mL (range, 1970-6000 mL). After automatic wash, the total CIK cell recovery was 85.3 percent (range, 78.5%-97.5%), and living cells were greater than 95 percent. After thawing, the median recoveries of total nucleated cells and natural killer T (NKT) cells were, respectively, 80.7 percent (range, 65%-95.5%) and 90.5 percent (range, 70.5%-98.5%). Thawed cells preserved their cytotoxic activity after cryopreservation (approx. 50% lysis at effector:target ratio of 40:1). CONCLUSION: The automatic wash with the CytoMate showed a good recovery of viable CIK cells during expansion and allowed an efficient manipulation of thawed cells. The use of this simple and efficient washing technique is suitable for clinical-grade processing in cellular therapy protocols.