Laser ablation-inductively coupled plasma mass spectrometry: an emerging technology for detecting rare cells in tissue sections.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Clinical management of patients receiving cell-based immunoregulatory therapy.

Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Regulatory macrophages as therapeutic targets and therapeutic agents in solid organ transplantation.

PURPOSE OF REVIEW: This review aims to provide a basic introduction to human macrophage biology and an appreciation of the diverse roles played by macrophage subsets in allograft damage and repair. Current and future strategies for therapeutically manipulating macrophage behaviour are discussed. RECENT FINDINGS: Macrophages are extremely versatile effector cells that exert both immunostimulatory and immunosuppressive effects. This adaptability cannot be explained by differentiation into committed sublineages, but instead reflects the ability of macrophages to rapidly transition between states of functional polarisation. Consequently, categorisation of macrophage subpopulations is not straightforward and this, in turn, creates difficulties in studying their pathophysiology. Nevertheless, particular macrophage subpopulations have been implicated in exacerbating or attenuating ischaemia-reperfusion injury, rejection reactions and allograft fibrosis. Three general strategies for therapeutically targeting macrophages can be envisaged, namely, depletional approaches, in-situ repolarisation towards a regulatory or tissue-reparative phenotype, and ex-vivo generation of regulatory macrophages (M reg) as a cell-based therapy. SUMMARY: As critical determinants of the local and systemic immune response to solid organ allografts, macrophage subpopulations represent attractive therapeutic targets. Rapid progress is being made in the implementation of novel macrophage-targeted therapies, particularly in the use of ex-vivo-generated M regs as a cell-based medicinal product.

Laser Ablation Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Multiparametric Analysis of Tissue Antigens.

New analytical techniques for multiparametric characterisation of individual cells are likely to reveal important information about the heterogeneity of immunological responses at the single-cell level. In this proof-of-principle study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was applied to the problem of concurrently detecting 24 lineage and activation markers expressed by human leucocytes. This approach was sufficiently sensitive and specific to identify subpopulations of isolated T, B, and natural killer cells. Leucocyte subsets were also accurately detected within unfractionated peripheral blood mononuclear cells preparations. Accordingly, we judge LA-ICP-MS to be a suitable method for assessing expression of multiple tissue antigens in solid-phase biological specimens, such as tissue sections, cytospins, or cells grown on slides. These results augur well for future development of LA-ICP-MS-based bioimaging instruments for general users.

In question: the scientific value of preclinical safety pharmacology and toxicology studies with cell-based therapies.

A new cell-based medicinal product containing human regulatory macrophages, known as Mreg_UKR, has been developed and conforms to expectations of a therapeutic drug. Here, Mreg_UKR was subjected to pharmacokinetic, safety pharmacology, and toxicological testing, which identified no adverse reactions. These results would normally be interpreted as evidence of the probable clinical safety of Mreg_UKR; however, we contend that, owing to their uncertain biological relevance, our data do not fully support this conclusion. This leads us to question whether there is adequate scientific justification for preclinical safety testing of similar novel cell-based medicinal products using animal models. In earlier work, two patients were treated with regulatory macrophages prior to kidney transplantation. In our opinion, the absence of acute or chronic adverse effects in these cases is the most convincing available evidence of the likely safety of Mreg_UKR in future recipients. On this basis, we consider that safety information from previous clinical investigations of related cell products should carry greater weight than preclinical data when evaluating the safety profile of novel cell-based medicinal products. By extension, we argue that omitting extensive preclinical safety studies before conducting small-scale exploratory clinical investigations of novel cell-based medicinal products data may be justifiable in some instances.