Managing leukapheresis in adult and pediatric patients eligible for chimeric antigen receptor T-cell therapy: suggestions from an Italian Expert Panel.

Chimeric antigen receptor (CAR) T-cell therapy relies on T cells engineered to target specific tumor antigens such as CD-19 in B-cell malignancies. In this setting, the commercially available products have offered a potential long-term cure for both pediatric and adult patients. Yet manufacturing CAR T cells is a cumbersome, multistep process, the success of which strictly depends on the characteristics of the starting material, i.e., lymphocyte collection yield and composition. These, in turn, might be affected by patient factors such as age, performance status, comorbidities, and previous therapies. Ideally, CAR T-cell therapies are a one-off treatment; therefore, optimization and the possible standardization of the leukapheresis procedure is critical, also in view of the novel CAR T cells currently under investigation for hematological malignancies and solid tumors. The most recent Best Practice recommendations for the management of children and adults undergoing CAR T-cell therapy provide a comprehensive guide to their use. However, their application in local practice is not straightforward and some grey areas remain. An Italian Expert Panel of apheresis specialists and hematologists from the centers authorized to administer CAR T-cell therapy took part in a detailed discussion on the following: 1) pre-apheresis patient evaluation; 2) management of the leukapheresis procedure, also in special situations represented by low lymphocyte count, peripheral blastosis, pediatric population <25 kg, and the COVID-19 outbreak; and 3) release and cryopreservation of the apheresis unit. This article presents some of the important challenges that must be faced to optimize the leukapheresis procedure and offers suggestions as to how to improve it, some of which are specific to the Italian setting.

Increased production of soluble HLA-G molecules in stimulated peripheral blood mononuclear cells following extracorporeal photopheresis: is it a mechanism involved in the therapeutic effect of the procedure?

We hypothesized that the effects of extracorporeal photopheresis (ECP) are mediated by induction of immunosuppressive cytokines like IL-10, which enhances synthesis of HLA-G molecules. HLA-G products are expressed by CD14+ peripheral blood mononuclear cells (PBMC) and play an important role in inhibition of cell mediated immunity. ECP induces apoptosis in lymphocytes but not in CD14+ cells. We, therefore, investigated the concentrations both of IL-10 and of soluble HLA-G5/sHLA-G1 molecules in supernatants from cultures of lipopolysaccharide-stimulated PBMC obtained from leukocyte collection bags of 10 patients receiving ECP for graft versus host disease both before (pre-irradiation) and after (post-irradiation) exposure to 8-methoxypsoralen and UVA irradiation. Levels of both IL-10 and HLA-G5/sHLA-G1 molecules were increased in the post-irradiation cultures. This suggests that therapeutic effects of ECP could be mediated by increased production of IL-10 and tolerogenic HLA-G molecules.

Caspase-9 is the upstream caspase activated by 8-methoxypsoralen and ultraviolet-A radiation treatment of Jurkat T leukemia cells and normal T lymphocytes.

BACKGROUND AND OBJECTIVES: A combination of 8-methoxypsoralen and ultraviolet-A radiation (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however scarce information is available concerning the apoptotic pathways activated by PUVA. DESIGN AND METHODS: We used Jurkat human T leukemia cells and normal T lymphocytes to analyze the PUVA-triggered caspase activation pattern by means of immunoblot analysis, in vitro caspase activity assays, and selective caspase inhibitors coupled to flow cytometric analysis. RESULTS: PUVA treatment induced activation of apical caspases-9 and -8, and of effector caspases-3 and -7 in Jurkat cells and human T lymphocytes. While activation of caspase-9 occurred as early as 1 h after PUVA treatment of Jurkat cells, procaspase-8 cleavage was delayed and was detected 6 h after the exposure. Also in normal T lymphocytes, cleavage of caspase-8 was subsequent to activation of caspase-9. PUVA-dependent proteolytic cleavage of procaspase-8 was blocked by inhibitors selective for either caspase-9 or -3. Moreover, procaspase-8 was cleaved in vitro by activated caspase-3, which gave rise to proteolytic fragments equivalent to those generated in vivo. INTERPRETATION AND CONCLUSIONS: Activation of caspase-8 in PUVA-treated Jurkat cells and normal T lymphocytes is secondary to up-regulation of caspase-9. Overall, our results identify caspase-9 as the critical upstream caspase initiating apoptosis by PUVA in Jurkat T-cells and human T lymphocytes.