Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells.

BACKGROUND: The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum. METHODS: T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity. RESULTS: CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+. CONCLUSION: Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product.

Differential activation of cord blood and peripheral blood natural killer cells by cytokines.

BACKGROUND AIMS: Natural killer (NK) cells play important roles in the clearance of infection and transformed cells. Cord blood (CB) is currently used as a source of hematopoietic stem cells for transplantation and is a potential source of NK cells for immunotherapy. We previously showed that CB NK cells are immature and less cytotoxic as compared with peripheral blood (PB) NK cells. We aimed to identify which cytokines, among interleukin (IL)-2, IL-12, IL-15 and IL-18 and their combinations, could fully activate CB NK cells as compared with PB NK cells. METHODS: We performed a comprehensive analysis of phenotype and functionality of cytokine-activated NK cells. RESULTS: Our results show that the lower responsiveness of CB NK cells to IL-2 is associated with lower levels of expression of IL-2 receptors and decreased phosphorylation of STAT5 as compared with PB NK cells. Activation of CB NK cells with IL-15+18 led to the most robust proliferative response and higher interferon-γ and tumor necrosis factor-α secretion, whereas activation with IL-15+2 promoted enhanced cytotoxicity. PB NK cells responded significantly better to IL-2 than to CB NK cells but were also fully activated with other cytokine treatments including IL-15, IL-15+2 or IL-15+18. It was also possible to use cytokines to generate memory-like NK cells, with sustained ability to produce interferon-γ, from both CB and PB. CONCLUSIONS: CB NK cells are fully functional on activation with IL-15+2 or IL-15+18 rather than IL-2 alone as observed for PB NK cells. These cytokines should be considered in the future to activate CB NK cells for therapeutic purposes.

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2.

BACKGROUND AIMS: Graft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts. METHODS: An approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2. RESULTS: MMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells. CONCLUSIONS: This study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.

The unique profile of cord blood natural killer cells balances incomplete maturation and effective killing function upon activation.

Cord blood (CB) is increasingly used as a source of stem cells for hematopoietic stem cell transplantation, and natural killer (NK) cells may be the effectors of the antileukemic response observed after CB transplantation. Here, we analyzed the phenotype and functions of CB NK cell subsets. We determined that the percentage of NK cells was higher in CB compared with peripheral blood (PB). Furthermore, there was a higher percentage of the CD56(bright) subset in CB. CB NK cells reached a late stage of differentiation, but exhibited higher expression of NKG2A and expressed fewer killer-cell immunoglobulin-like receptors, suggesting an incomplete maturation. CB NK cells highly expressed CXCR4, but did not express L-selectin, highlighting unique homing properties of CB NK cells. CB NK cells proliferated in response to interleukin-2 and degranulated in response to stimulation with tumor cells, but failed to lyse K562 cells in (51)Cr-release assay. CB NK cells exhibited a lower interferon-γ production in comparison with PB NK cells. Culture with IL-2 increased CB NK cell functions. Our study sheds light on CB NK cell properties and highlights the potential of CB as a source of NK cells for immunotherapy.