Peripheral blood mononuclear cell collection for immunological investigation of chronic graft-versus-host disease: safety and efficacy of leukapheresis in 132 patients | Journal of Translational Medicine

Peripheral blood mononuclear cell collection for immunological investigation of chronic graft-versus-host disease: safety and efficacy of leukapheresis in 132 patients |  Journal of Translational Medicine

Advances in reduced-intensity conditioning regimens and more effective antimicrobials have significantly reduced early post-transplant-related morbidity and mortality after allo-HCT. However, late post-transplant morbidity and mortality, largely due to chronic GVHD, remain a challenge. [18] The pathophysiology of chronic GVHD is complex and a better understanding of it is essential to advance its management.

Steady-state peripheral blood leukapheresis or peripheral blood MNC collection for extracorporeal photopheresis (ECP) have been used therapeutically in chronic GVHD. [19,20,21,22]. Similarly, lymphapheresis is the first step in the chimeric antigen receptor (CAR) T-cell manufacturing process and has been a safe and reliable procedure for the collection of CD3+ lymphocytes from patients after allogeneic HCT. [23, 24]. These procedures require a sufficient number of cells for therapeutic processing, which could be a significant challenge in chronic GVHD, where patients are often frail or lymphopenic. The routine use of leukapheresis to acquire larger numbers of MNC solely for research purposes in chronic GVHD has not been done due to concerns of logistics, feasibility, and patient safety. This current study provides evidence for the use of leukapheresis as a safe and powerful research tool to advance knowledge about chronic GVHD and provides benchmarks for developing novel therapeutic interventions, such as regulatory T-cell infusion for GVHD. [25] or CAR T cell therapy [26, 27].

One hundred thirty-two (40%) of the chronic GVHD patients in this study underwent investigational leukapheresis. The results show that regardless of disease severity, extent of sclerotic disease, age, or blood cell counts, steady-state PBMC leukapheresis is a safe, well-tolerated, and overall effective method for collection. of PBMC on a large scale. 71% achieved the 2 × 10^9 PBMC collection goal, despite our GVHD population being enriched for moderate/severe disease (97%), including many with profound sclerosis (40%). In particular, 48 patients (37%) were lymphopenic and had a pre-leukapheresis ALC < 1.0 K/µl and would likely be very difficult to study for conventional (20–50 ml) or large investigational blood immunoassays. volume (250 ml). raffles. No patients experienced grade ≥ 2 adverse events due to leukapheresis, and all adverse events resolved by discharge from the apheresis unit.

This report demonstrates the feasibility and safety of large-scale collection and storage of PMNC for research purposes and should support such practice in IRB-approved chronic GVHD research protocols. As an example (see Figs. 3 and 4), if the research needs to use B and/or T lymphocytes and/or monocytes, depending on the specific needs of the project, it might be possible to set an arbitrary minimum collection target lymphocytes/MNC in the apheresis bag, for example, 300 × 10e6, or 500 × 10e6, or 1.0 × 10e9 per apheresis bag. Thus, even for the patients/procedures with the lowest pre-apheresis ALC count, a minimum of 600 × 10e6 MNC was collected in the apheresis bag, which should be adequate for routine research trials (Fig. . two). Looking specifically at total apheresis yield and the lower range of MNC yields, 14 of 132 collections (11%) had yields of < 1.0 × 10e9 (1000 × 10e6), only 3 (2.3%) had yields of < 0.5 × 10e9 (500 × 10e6), and only 1 had yields < 0.3 × 10e9 (or 300 × 10e6), depending on what would be considered a minimum threshold for a successful or usable collection. As an example of its use in research, we and our NIH collaborators were able to separate adequate numbers of FACS-selected cell populations for cell culture and gene expression studies related to mechanisms of chronic GVHD. [6]. Similarly, the availability of large numbers of lymphocytes using 2 L leukapheresis allowed collaborators to analyze gene expression in cultures of sorted B cells from patients with chronic GVHD; these studies identified a mechanistic link between NOTCH2 activation and robust B-cell activation in chronic GVHD [28]. On the other hand, these yields may be suboptimal in the clinical setting, that is, for therapeutic purposes. For example, CAR T cell protocols typically specify a much higher minimum number of CD3+ cells in the apheresis bag (eg, a minimum of 1,000 × 10e6 or 1.0 × 10e9, to have at least 2 aliquots of CD3+ cells). 300–500 × 10e6 CD3+ per aliquot for culture).

Chronic GVHD patients often have decreased peripheral blood cell counts, including anemia, thrombocytopenia, neutropenia, and particularly lymphopenia, due to systemic immunosuppressive treatments or the chronic GVHD process itself. Only mild decreases in peripheral counts were seen in this study after investigational leukapheresis, and no patients required transfusion of blood products, colony-stimulating factors, or prophylactic antibiotics. However, in-person follow-up after the procedure was limited as the leukapheresis was performed on the last day of their outpatient week and patients returned home a short time later. All patients received a follow-up phone call from a study doctor one week after returning home, so late-onset or longer-lasting adverse events may not have been adequately captured. Another limitation of our study could be the fact that our patients’ procedures were performed between 2005 and 2014, and both the CS300 and Spectra instruments have since been withdrawn. There are newer devices like Amicus and Optia. Experience with healthy volunteers in our clinic shows a lower overall platelet yield with Amicus and Optia. For the healthy volunteer cohort, monocyte collection efficiency was lower with the CS-3000 than with any of the other devices (see Table A, Supplementary Materials).

Advancing efficacy and developing personalized approaches to the treatment of chronic GVHD remains a significant unmet need to improve long-term outcomes for patients undergoing allogeneic hematopoietic cell transplantation. [29]. Despite great advances in understanding the biology of chronic GVHD, more progress is needed. A better understanding of clinical-biological correlations is needed. Concerns about logistics, feasibility, and safety have been a major barrier to performing research-directed leukapheresis to reliably obtain large amounts of PBMNC during clinical studies of chronic GVHD. Since approximately one-third of patients with chronic GVHD have substantial lymphopenia, many patients, presumably those with more severe disease [12]are excluded from immunological research studies using conventional peripheral blood collection techniques.

This study conclusively demonstrates the feasibility and safety of large-scale PMNC collection and storage for research purposes and should support such practice in IRB-approved chronic GVHD research protocols.

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