Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy.

BACKGROUND: The wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons for and solutions to this failure, taking as our model the preparation of Epstein-Barr virus (EBV)-specific T cells (EBVSTs) for the treatment of EBV-positive lymphoma. EBVSTs could not be manufactured from almost one-third of patients, either because they failed to expand, or they expanded, but lacked EBV specificity. We identified an underlying cause of this problem and established a clinically feasible approach to overcome it. METHODS: CD45RO+CD45RA- memory compartment residing antigen-specific T cells were enriched by depleting CD45RA positive (+) peripheral blood mononuclear cells (PBMCs) that include naïve T cells, among other subsets, prior to EBV antigen stimulation. We then compared the phenotype, specificity, function and T-cell receptor (TCR) Vß repertoire of EBVSTs expanded from unfractionated whole (W)-PBMCs and CD45RA-depleted (RAD)-PBMCs on day 16. To identify the CD45RA component that inhibited EBVST outgrowth, isolated CD45RA+ subsets were added back to RAD-PBMCs followed by expansion and characterization. The in vivo potency of W-EBVSTs and RAD-EBVSTs was compared in a murine xenograft model of autologous EBV+ lymphoma. RESULTS: Depletion of CD45RA+ PBMCs before antigen stimulation increased EBVST expansion, antigen-specificity and potency in vitro and in vivo. TCR sequencing revealed a selective outgrowth in RAD-EBVSTs of clonotypes that expanded poorly in W-EBVSTs. Inhibition of antigen-stimulated T cells by CD45RA+ PBMCs could be reproduced only by the naïve T-cell fraction, while CD45RA+ regulatory T cells, natural killer cells, stem cell memory and effector memory subsets lacked inhibitory activity. Crucially, CD45RA depletion of PBMCs from patients with lymphoma enabled the outgrowth of EBVSTs that failed to expand from W-PBMCs. This enhanced specificity extended to T cells specific for other viruses. CONCLUSION: Our findings suggest that naïve T cells inhibit the outgrowth of antigen-stimulated memory T cells, highlighting the profound effects of intra-T-cell subset interactions. Having overcome our inability to generate EBVSTs from many patients with lymphoma, we have introduced CD45RA depletion into three clinical trials: NCT01555892 and NCT04288726 using autologous and allogeneic EBVSTs to treat lymphoma and NCT04013802 using multivirus-specific T cells to treat viral infections after hematopoietic stem cell transplantation.

Histone induced platelet aggregation is inhibited by normal albumin.

INTRODUCTION: Histones are small, nuclear proteins that serve to package DNA. Recent reports suggest that extracellular histones, including histone H4, may contribute to the pathogenesis of sepsis; they promote platelet aggregation and thrombosis when released into the circulation during inflammation or cell death. The mechanisms by which the body minimizes the deleterious effects of circulating histones are unclear. Because histones can bind to plasma proteins, including albumin, we hypothesized that normal albumin can prevent histones from activating platelets. MATERIALS AND METHODS: Platelets and platelet-free plasma were obtained from healthy, adult subjects. The dose-dependent effects of histone H4 on platelet aggregation were studied by optical aggregometry. The effects of native and albumin-depleted plasma (prepared by affinity chromatography) on histone-induced platelet aggregation were also assessed. The effects of normal and surface-neutralized albumin (through modification of carboxyl groups) on histone-induced platelet activation and aggregation were evaluated using flow cytometry and aggregometry. RESULTS: Histone H4 induced platelet aggregation in a dose-dependent manner. This histone-induced platelet aggregation was inhibited by both plasma and human serum albumin in a dose-dependent fashion. Furthermore, depletion of albumin from plasma reduced its ability to inhibit aggregation. Finally, surface neutralization of albumin decreased its ability to inhibit histone-induced activation and aggregation. DISCUSSION: These data suggest that normal albumin serves a role in preventing histone-induced platelet aggregation in a charge-dependent manner.