Single-cell multiomic dissection of response and resistance to chimeric antigen receptor T cells against BCMA in relapsed multiple myeloma.

Markers that predict response and resistance to chimeric antigen receptor (CAR) T cells in relapsed/refractory multiple myeloma are currently missing. We subjected mononuclear cells isolated from peripheral blood and bone marrow before and after the application of approved B cell maturation antigen-directed CAR T cells to single-cell multiomic analyses to identify markers associated with resistance and early relapse. Differences between responders and nonresponders were identified at the time of leukapheresis. Nonresponders showed an immunosuppressive microenvironment characterized by increased numbers of monocytes expressing the immune checkpoint molecule CD39 and suppressed CD8+ T cell and natural killer cell function. Analysis of CAR T cells showed cytotoxic and exhausted phenotypes in hyperexpanded clones compared to low/intermediate expanded clones. We identified potential immunotherapy targets on CAR T cells, like PD1, to improve their functionality and durability. Our work provides evidence that an immunosuppressive microenvironment causes resistance to CAR T cell therapies in multiple myeloma.

Cellular dynamics following CAR T cell therapy are associated with response and toxicity in relapsed/refractory myeloma.

B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, data on cellular (CAR) T cell dynamics and the association with response, resistance or the occurrence of cytokine release syndrome (CRS) are limited. Therefore, we performed a comprehensive flow cytometry analysis of 27 RRMM patients treated with Idecabtagene vicleucel (Ide-cel) to assess the expansion capacity, persistence and effects on bystander cells of BCMA-targeting CAR T cells. Additionally, we addressed side effects, like cytokine release syndrome (CRS) and cytopenia. Our results show that in vivo expansion of CD8+ CAR T cells is correlated to response, however persistence is not essential for durable remission in RRMM patients. In addition, our data provide evidence, that an increased fraction of CD8+ T cells at day of leukapheresis in combination with successful lymphodepletion positively influence the outcome. We show that patients at risk for higher-grade CRS can be identified already prior to lymphodepletion. Our extensive characterization contributes to a better understanding of the dynamics and effects of BCMA-targeting CAR T cells, in order to predict the response of individual patients as well as side effects, which can be counteracted at an early stage or even prevented.

Extracellular Vesicles of Mesenchymal Stromal Cells Can be Taken Up by Microglial Cells and Partially Prevent the Stimulation Induced by ß-amyloid.

Mesenchymal stromal/stem cells (MSCs) have great capacity for immune regulation. MSCs provide protective paracrine effects, which are partially exerted by extracellular vesicles (EVs). It has been reported that MSCs-derived EVs (MSC-EVs) contain soluble factors, such as cytokines, chemokines, growth factors and even microRNAs, which confer them similar anti-inflammatory and regenerative effects to MSCs. Moreover, MSCs modulate microglia activation through a dual mechanism of action that relies both on cell contact and secreted factors. Microglia cells are the central nervous system immune cells and the main mediators of the inflammation leading to neurodegenerative disorders. Here, we investigated whether MSC-EVs affect the activation of microglia cells by ß-amyloid aggregates. We show that the presence of MSC-EVs can prevent the upregulation of pro-inflammatory mediators such as tumor necrosis factor (TNF)-α and nitric oxide (NO). Both are up-regulated in neurodegenerative diseases representing chronic inflammation, as in Alzheimer's disease. We demonstrate that MSC-EVs are internalized by the microglia cells. Further, our study supports the use of MSC-EVs as a promising therapeutic tool to treat neuroinflammatory diseases.Significance StatementIt has been reported that mesenchymal stromal/stem cells and MSC-derived small extracellular vesicles have therapeutic effects in the treatment of various degenerative and inflammatory diseases. Extracellular vesicles are loaded with proteins, lipids and RNA and act as intercellular communication mediators. Here we show that extracellular vesicles can be taken up by murine microglial cells. In addition, they partially reduce the activation of microglial cells against ß-amyloid aggregates. This inhibition of microglia activation may present an effective strategy for the control/therapy of neurodegenerative diseases such as Alzheimer's disease.

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells.

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are "label-free", while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

Laboratory Workup of Lymphoma in Adults: Guideline From the American Society for Clinical Pathology and the College of American Pathologists.

CONTEXT.­: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery led to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. OBJECTIVE.­: To develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. DESIGN.­: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were derived based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. RESULTS.­: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS.­: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions on specimen suitability, diagnostic capabilities, and correct use of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Laboratory Workup of Lymphoma in Adults.

OBJECTIVES: The diagnostic workup of lymphoma continues to evolve rapidly as experience and discovery lead to the addition of new clinicopathologic entities and techniques to differentiate them. The optimal clinically effective, efficient, and cost-effective approach to diagnosis that is safe for patients can be elusive, in both community-based and academic practice. Studies suggest that there is variation in practice in both settings. THE AIM OF THIS REVIEW IS TO: develop an evidence-based guideline for the preanalytic phase of testing, focusing on specimen requirements for the diagnostic evaluation of lymphoma. METHODS: The American Society for Clinical Pathology, the College of American Pathologists, and the American Society of Hematology convened a panel of experts in the laboratory workup of lymphoma to develop evidence-based recommendations. The panel conducted a systematic review of the literature to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were derived based on the available evidence, the strength of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS: Thirteen guideline statements were established to optimize specimen selection, ancillary diagnostic testing, and appropriate follow-up for safe and accurate diagnosis of indolent and aggressive lymphoma. CONCLUSIONS: Primary diagnosis and classification of lymphoma can be achieved with a variety of specimens. Application of the recommendations can guide decisions about specimen suitability, diagnostic capabilities, and correct utilization of ancillary testing. Disease prevalence in patient populations, availability of ancillary testing, and diagnostic goals should be incorporated into algorithms tailored to each practice environment.

Nicotinamide Inhibits Self-renewal and Induces Granulocyte Differentiation of Multipotent Progenitor Cells.

Nicotinamide (NAM) a form of vitamin B3, is an essential precursor of NAD. This dinucleotide (pyridine nucleotide) participates in the regulation of fundamental processes including transcription, cell cycle progression and DNA repair. Here we assessed the effect of NAM on myeloid differentiation of the IL-3 dependent, multipotent hematopoietic progenitor cell line FDCP-Mix. We found that NAM reduces the pSTAT5 signaling response, cell cycling and self-renewal potential. It initiates an atypical program of myeloid differentiation that results in the emergence of granulocytic cells in the absence of added myeloid differentiation factors. NAM did not affect the expression the of cell surface granulocyte marker GR1 but led to a strong downregulation of MHC-II molecules. Taken together our data show that NAM induces a differentiation program in hematopoietic progenitors prompting them to undergo differentiation along the granulocyte path without reaching the status of fully developed granulocytes. Graphical abstract.

3D Scaffold-Based Macrophage Fibroblast Coculture Model Reveals IL-10 Dependence of Wound Resolution Phase.

Persistent inflammation and impaired repair in dermal wound healing are frequently associated with cell-cell and cell-matrix miscommunication. A direct coculture model of primary human myofibroblasts (MyoFB) and M-CSF-differentiated macrophages (M-Mɸ) in fibrillar three-dimensional Collagen I (Coll I) matrices is developed to study intercellular interactions. The coculture experiments reveal the number of M-Mɸ regulated MyoFB dedifferentiation in a dose-dependent manner. The amount of MyoFB decreases in dependence of the number of cocultured M-Mɸ, even in the presence of MyoFB-inducing transforming growth factor ß1 (TGF-ß1 ). Gene expression analysis of matrix proteins (collagen I, collagen III, ED-A-fibronectin) confirms the results of an altered MyoFB phenotype. Additionally, M-Mɸ is shown to be the main source of secreted cytokine interleukin-10 (IL-10), which is suggested to affect MyoFB dedifferentiation. These findings indicate a paracrine impact of IL-10 secretion by M-Mɸ on the MyoFB differentiation status counteracting the TGF-ß1 -driven MyoFB activation. Hence, the in vitro coculture model simulates physiological situations during wound resolution and underlines the importance of paracrine IL-10 signals by M-Mɸ. In sum, the 3D Coll I-based matrices with a MyoFB-M-Mɸ coculture form a highly relevant biomimetic model of late stages of wound healing.

NAD metabolites interfere with proliferation and functional properties of THP-1 cells.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

CD14 Counterregulates Lipopolysacharide-Induced Tumor Necrosis Factor-α Production in a Macrophage Subset.

In response to GM-CSF or M-CSF, macrophages (MΦ) can acquire pro- or anti-inflammatory properties, respectively. Given the importance of CD14 and Toll-like receptor (TLR) 4 in lipopolysaccharide (LPS)-induced signaling, we studied the effect of anti-CD14 antibody mediated CD14 blockade on LPS-induced cytokine production, signal transduction and on the expression levels of CD14 and TLR4 in GM-MΦ and M-MΦ. We found M-MΦ to express higher levels of both surface antigens and to produce more interferon (IFN)-ß and interleukin-10, but less tumor necrosis factor (TNF)-α than GM-MΦ. Blockage of CD14 at high LPS concentrations increased the production of proinflammatory cytokines and decreased that of IFN-ß in M-MΦ but not in GM-MΦ. We show that phosphorylation states of signaling molecules of the MyD88 (myeloid differentiation primary response 88), TRIF (TIR-domain-containing adapter-inducing IFN-ß) and MAPK (mitogen-activated protein kinase) pathways are not altered in any way that would account for the cytokine overshoot reaction. However, CD14 blockage in M-MΦ decreased TLR4 and CD14 expression levels, regardless of the presence of LPS, indicating that the loss of the surface molecules prevented LPS from initiating TRIF signaling. As TNF-α synthesis was even upregulated under these experimental conditions, we suggest that TRIF is normally involved in restricting LPS-induced TNF-α overproduction. Thus, surface CD14 plays a decisive role in the biological response by determining LPS-induced signaling.

A comparative study of two separation methods to isolate monocytes.

Separation of specific blood cells is necessary for a deeper insight into their role in health and disease. To obtain such cells, efficient and robust isolation methods are needed. We compare here the Fab-based Traceless Affinity Cell Selection (TACS®) technology and the Magnetic Activated Cell Sorting (MACS®) technology to isolate human monocytes from whole blood and buffy coats as well as the differentiation of the isolated monocytes to dendritic cells (DCs). TACS® is a positive selection technology using immune affinity chromatography based on CD-specific low affinity Fab-fragments for the reversible capture and release of target cells. The positive selection by MACS® is based on magnetic beads coated with specific high affinity monoclonal antibodies to catch the target cells. The target cells separated by TACS® are "label-free" while cells positively isolated by MACS® will carry the cell specific label. Our data show that the separation methods described here are well suited to obtain functional monocytes of high quality and purity. A differentiation of the cells into DCs leads to comparable results with the exception that CD1a expression levels on immature and mature DCs are elevated when monocytes are isolated using the TACS® technology. Taken together, our results suggest that the TACS® method may be of advantage when preparing monocytes and monocyte-derived DCs for functional analyses, while the MACS® method seems to be capable of higher monocyte recoveries. © 2018 International Society for Advancement of Cytometry.

Reliability and Validity of Proposed Risk Stratification Methods for Laboratory Developed Tests.

OBJECTIVES: To determine whether different laboratory developed test (LDT) risk stratification proposals would assign differing levels of risk to selected LDTs as a measure of the validity of those proposals, and whether there would be differing interrater agreement rates as a measure of the reliability of those proposals. METHODS: A total of 4 reviewers applied 6 proposals for risk stratification of 4 LDTs. Interrater agreement was calculated as a measure of the reliability of the proposals. Also, a consensus risk categorization and concordance rate for each LDT was developed as a measure of the validity of the proposals. RESULTS: Interrater agreement rates (reliability) ranged from 38% to 100%, and concordance rates (validity) ranged from 20% to 100%. CONCLUSIONS: A spectrum of reliability and validity was observed depending on the policy used and the LDT categorized. Before implementation or legislation of risk-stratification methods, large evaluations of reliability and validity should be conducted on any proposed method.

Facing the Inevitable: Being Prepared for Regulatory Requirements for Laboratory Developed Tests.

OBJECTIVES: We introduce regulatory terms, definitions, and the Quality System Regulation as proposed by the US Food and Drug Administration in the 2014 draft guidance entitled Framework for Regulatory Oversight of Laboratory Developed Tests and explore medical device requirements applicable to a laboratory environment to design, develop, and validate laboratory developed tests (LDTs). METHODS: We performed nine interviews with laboratory professionals to explore concerns and challenges regarding the draft, translated the results into operational factors, and surveyed professionals to test the factors that would comprise a regulatory quality management system framework. RESULTS: Nine interviewees and 35 survey respondents shared concerns of risk classification, process validation, patient safety, and general ambiguity regarding the proposed requirements for development of LDTs. CONCLUSIONS: Respondents agree that a regulatory quality management system is needed in laboratories that develop LDTs, but the translation and method for design control to a clinical laboratory do not exist. As a result, laboratories are taking the wait-and-see approach.

Molecular mechanism of LPS-induced TNF-α biosynthesis in polarized human macrophages.

In response to environmental stimuli such as granulocyte-macrophage or macrophage colony stimulating factor (GM-CSF/M-CSF), macrophages (MΦ) can acquire distinct functional phenotypes that control inflammatory processes on the one hand and contribute to a broad spectrum of pathologies on the other. Potential intervention strategies will require an understanding of the signalling processes that are associated with macrophage polarization. In the present study, we show that M-MΦ produce more IFN-ß and IL-10 and a lot less TNF-α than do GM-MΦ in response to LPS. To define the molecular mechanisms that underlie the biosynthesis of TNF-α we carried out a detailed investigation of the LPS-induced activation of the canonical and non-canonical myeloid differentiation primary response 88 (MyD88)-dependent signal transduction pathways as well as the TIR-domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathway. Our results show that all three pathways are activated in both cell types and that the activation is more pronounced in M-MΦ. While IL-10 was found to interfere with TNF-α production in M-MΦ, we exclude a decisive role for IFN-ß in this respect. Furthermore, we demonstrate that TNF-α mRNA is markedly destabilized in M-MΦ and that expression of the mRNA destabilizing protein tristetraprolin is greatly enhanced in these cells. Collectively, our study suggests that differential effects of LPS on TNF-α mRNA turnover and on signal transduction pathways influence the amount of TNF-α finally produced by GM-MΦ and M-MΦ.

Laboratory-Developed Tests: A Legislative and Regulatory Review.

BACKGROUND: Twenty-five years ago, the Food and Drug Administration (FDA) asserted in a draft document that "home brew" tests-now commonly referred to as laboratory-developed tests (LDTs)-are subject to the same regulatory oversight as other in vitro diagnostics (IVDs)4. In 2010, the FDA began work on developing a proposed framework for future LDT oversight. Released in 2014, the draft guidance sparked an intense debate over potential LDT regulation. While the proposed guidance has not been implemented, many questions regarding LDT oversight remain unresolved. CONTENT: This review provides an overview of federal statutes and regulations related to IVDs and clinical laboratory operations, with a focus on those potentially applicable to LDTs and proposed regulatory efforts. Sources reviewed include the Code of Federal Regulations, the Federal Register, congressional hearings, guidance and policy documents, position statements, published literature, and websites. SUMMARY: Federal statutes regarding IVDs were passed without substantive evidence of congressional consideration toward the concept of LDTs. The FDA has clear oversight authority over IVD reagents introduced into interstate commerce. A 16-year delay in publicly asserting FDA authority over LDTs, the pursuit of a draft guidance approach toward oversight, and establishment of regulations under the Clinical Laboratory Improvement Amendments of 1988 (CLIA'88) applicable to LDTs contributed to community uncertainty toward LDT oversight. Future regulatory and/or legislative efforts may be required to resolve this uncertainty.

Teaching Laboratory Management Principles and Practices Through Mentorship and Graduated Responsibility: The Assistant Medical Directorship.

With the changing landscape of medicine in general, and pathology in particular, a greater emphasis is being placed on laboratory management as a means of controlling spiraling medical costs and improving health-care efficiency. To meet this challenge, pathology residency programs have begun to incorporate formal laboratory management training into their curricula, using institutional curricula and/or online laboratory management courses offered by professional organizations. At the University of Utah, and its affiliated national reference laboratory, ARUP Laboratories, Inc, interested residents are able to supplement the departmental lecture-based and online laboratory management curriculum by participating in assistant medical directorship programs in one of several pathology subspecialty disciplines. The goals of many of the assistant medical directorship positions include the development of laboratory management skills and competencies. A survey of current and recent assistant medical directorship participants revealed that the assistant medical directorship program serves as an excellent means of improving laboratory management skills, as well as improving performance as a fellow and practicing pathologist.

Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features.