Non-viral expression of chimeric antigen receptors with multiplex gene editing in primary T cells.

Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through the knockout of genes of interest while knocking in synthetic receptors that can recognize cancer cells. Performing multiple rounds of gene transfer with precise genome editing, termed multiplexing, remains a key challenge, especially for non-viral delivery platforms. Here, we demonstrate the efficient production of primary human T cells incorporating the knockout of three clinically relevant genes (B2M, TRAC, and PD1) along with the non-viral transfection of a CAR targeting disialoganglioside GD2. Multiplexed knockout results in high on-target deletion for all three genes, with low off-target editing and chromosome alterations. Incorporating non-viral delivery to knock in a GD2-CAR resulted in a TRAC-B2M-PD1-deficient GD2 CAR T-cell product with a central memory cell phenotype and high cytotoxicity against GD2-expressing neuroblastoma target cells. Multiplexed gene-editing with non-viral delivery by CRISPR/Cas9 is feasible and safe, with a high potential for rapid and efficient manufacturing of highly potent allogeneic CAR T-cell products.

Leveraging a smartphone to perform time-gated luminescence measurements.

Empowered by advanced on-board sensors, high-performance optics packages and ever-increasing computational power, smartphones have democratized data generation, collection, and analysis. Building on this capacity, many platforms have been developed to enable its use as an optical sensing platform for colorimetric and fluorescence measurements. In this paper, we report the ability to enable a smartphone to perform laboratory quality time-resolved analysis of luminescent samples via the exploitation of the rolling shutter mechanism of the native CMOS imager. We achieve this by leveraging the smartphone's standard image capture applications, commercially available image analysis software, and housing the device within a UV-LED containing case. These low-cost modifications enable us to demonstrate the smartphone's analytical potential by performing tasks ranging from authentication and encryption to the interrogation of packaging, compounds, and physical phenomena. This approach underscores the power of repurposing existing technologies to extend the reach and inclusivity of scientific exploration, opening new avenues for data collection and analysis.

Halo-A Universal Fluorescence Reader Based Threat Agent Detection Platform-A Proof of Concept Study Using SARS-CoV-2 Assays.

Polymerase chain reaction (PCR) remains the gold standard in disease diagnostics due to its extreme sensitivity and specificity. However, PCR tests are expensive and complex, require skilled personnel and specialized equipment to conduct the tests, and have long turnaround times. On the other hand, lateral flow immunoassay-based antigen tests are rapid, relatively inexpensive, and can be performed by untrained personnel at the point of care or even in the home. However, rapid antigen tests are less sensitive than PCR since they lack the inherent target amplification of PCR. It has been argued that rapid antigen tests are better indicators of infection in public health decision-making processes to test, trace, and isolate infected people to curtail further transmission. Hence, there is a critical need to increase the sensitivity of rapid antigen tests and create innovative solutions to achieve that goal. Herein, we report the development of a low-cost diagnostic platform, enabling rapid detection of SARS-CoV-2 under field or at-home conditions. This platform (Halo™) is a small, highly accurate, consumer-friendly diagnostic reader paired with fluorescently labeled lateral flow assays and custom software for collection and reporting of results. The focus of this study is to compare the analytical performance of HaloTM against comparable tests that use either colloidal gold nanoparticles or fluorescence-based reporters in simulated nasal matrix and not in clinical samples. Live virus data has demonstrated limit of detection performance of 1.9 TCID50/test in simulated nasal matrix for the delta variant, suggesting that single-assay detection of asymptomatic SARS-CoV-2 infections may be feasible. Performance of the system against all tested SARS CoV-2 virus variants showed comparable sensitivities indicating mutations in SARS-CoV-2 variants do not negatively impact the assay.

Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo ß7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Human multipotent adult progenitor cells effectively reduce graft-vs-host disease while preserving graft-vs-leukemia activity.

Graft-vs-host disease (GvHD) limits successful outcomes following allogeneic blood and marrow transplantation (allo-BMT). We examined whether the administration of human, bone marrow-derived, multipotent adult progenitor cells (MAPCs™) could regulate experimental GvHD. The immunoregulatory capacity of MAPC cells was evaluated in vivo using established murine GvHD models. Injection of MAPC cells on day +1 (D1) and +4 (D4) significantly reduced T-cell expansion and the numbers of donor-derived, Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)-producing, CD4+ and CD8+ cells by D10 compared with untreated controls. These findings were associated with reductions in serum levels of TNFα and IFNγ, intestinal and hepatic inflammation and systemic GvHD as measured by survival and clinical score. Biodistribution studies showed that MAPC cells tracked from the lung and to the liver, spleen, and mesenteric nodes within 24 hours after injection. MAPC cells inhibited mouse T-cell proliferation in vitro and this effect was associated with reduced T-cell activation and inflammatory cytokine secretion and robust increases in the concentrations of Prostaglandin E2 (PGE2) and Transforming Growth Factor Beta (TGFß). Indomethacin and E-prostanoid 2 (EP2) receptor antagonism both reversed while EP2 agonism restored MAPC cell-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following allo-BMT abrogated the protective effects of MAPC cells. Importantly, MAPC cells had no effect on the generation cytotoxic T lymphocyte activity in vitro, and the administration of MAPC cells in the setting of leukemic challenge resulted in superior leukemia-free survival. Collectively, these data provide valuable information regarding the biodistribution and regulatory capacity of MAPC cells, which may inform future clinical trial design.

Human Wharton's Jelly-Derived Stem Cells Display a Distinct Immunomodulatory and Proregenerative Transcriptional Signature Compared to Bone Marrow-Derived Stem Cells.

Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive and trophic support functions. While MSCs from different sources frequently display a similar appearance in culture, they often show differences in their surface marker and gene expression profiles. Although bone marrow is considered the "gold standard" tissue to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from more easily accessible extra-embryonic tissues such as the umbilical cord. In this study, we defined the best way to isolate MSCs from the Wharton's jelly of the human umbilical cord (WJ-MSC) and assessed the mesenchymal and immunological phenotype of BM-MSC and WJ-MSC. Moreover, the gene expression profile of established WJ-MSC cultures was compared to two different bone marrow-derived stem cell populations (BM-MSC and multipotent adult progenitor cells or MAPC®). We observed that explant culturing of Wharton's jelly matrix is superior to collagenase tissue digestion for obtaining mesenchymal-like cells, with explant isolated cells displaying increased expansion potential. While being phenotypically similar to adult MSCs, WJ-MSC show a different gene expression profile. Gene ontology analysis revealed that genes associated with cell adhesion, proliferation, and immune system functioning are enriched in WJ-MSC. In vivo transplantation confirms their immune modulatory effect on T cells, similar to BM-MSC and MAPC. Furthermore, WJ-MSC intrinsically overexpress genes involved in neurotrophic support and their secretome induces neuronal maturation of SH-SY5Y neuroblastoma cells to a greater extent than BM-MSC. This signature makes WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated central nervous system disorders such as multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

Biomanufacturing for clinically advanced cell therapies.

The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Crosstalk with Inflammatory Macrophages Shapes the Regulatory Properties of Multipotent Adult Progenitor Cells.

Macrophages and microglia are key effector cells in immune-mediated neuroinflammatory disorders. Driving myeloid cells towards an anti-inflammatory, tissue repair-promoting phenotype is considered a promising strategy to halt neuroinflammation and promote central nervous system (CNS) repair. In this study, we defined the impact of multipotent adult progenitor cells (MAPC), a stem cell population sharing common mesodermal origin with mesenchymal stem cells (MSCs), on the phenotype of macrophages and the reciprocal interactions between these two cell types. We show that MAPC suppress the secretion of tumor necrosis factor alpha (TNF-α) by inflammatory macrophages partially through a cyclooxygenase 2- (COX-2-) dependent mechanism. In turn, we demonstrate that inflammatory macrophages trigger the immunomodulatory properties of MAPC, including an increased expression of immunomodulatory mediators (e.g., inducible nitric oxide synthase (iNOS) and COX-2), chemokines, and chemokine receptors. Macrophage-primed MAPC secrete soluble factors that suppress TNF-α release by macrophages. Moreover, the MAPC secretome suppresses the antigen-specific proliferation of autoreactive T cells and the T cell stimulatory capacity of macrophages. Finally, MAPC increase their motility towards secreted factors of activated macrophages. Collectively, these in vitro findings reveal intimate reciprocal interactions between MAPC and inflammatory macrophages, which are of importance in the design of MAPC-based therapeutic strategies for neuroinflammatory disorders in which myeloid cells play a crucial role.

Human factors analysis of the CardioQuick Patch®: A novel engineering solution to the problem of electrode misplacement during 12-lead electrocardiogram acquisition.

INTRODUCTION: The CardioQuick Patch® (CQP) has been developed to assist operators in accurately positioning precordial electrodes during 12-lead electrocardiogram (ECG) acquisition. This study describes the CQP design and assesses the device in comparison to conventional electrode application. METHODS: Twenty ECG technicians were recruited and a total of 60 ECG acquisitions were performed on the same patient model over four phases: (1) all participants applied single electrodes to the patient; (2) all participants were then re-trained on electrode placement and on how to use the CQP; (3) participants were randomly divided into two groups, the standard group applied single electrodes and the CQP group used the CQP; (4) after a one day interval, the same participants returned to carry out the same procedure on the same patient (measuring intra-practitioner variability). Accuracy was measured with reference to pre-marked correct locations using ultra violet ink. NASA-TLK was used to measure cognitive workload and the Systematic Usability Scale (SUS) was used to quantify the usability of the CQP. RESULTS: There was a large difference between the minimum time taken to complete each approach (CQP=38.58s vs. 65.96s). The standard group exhibited significant levels of electrode placement error (V1=25.35mm±29.33, V2=18.1mm±24.49, V3=38.65mm±15.57, V4=37.73mm±12.14, V5=35.75mm±15.61, V6=44.15mm±14.32). The CQP group had statistically greater accuracy when placing five of the six electrodes (V1=6.68mm±8.53 [p<0.001], V2=8.8mm±9.64 [p=0.122], V3=6.83mm±8.99 [p<0.001], V4=14.90mm±11.76 [p<0.001], V5=8.63mm±10.70 [p<0.001], V6=18.13mm±14.37 [p<0.001]). There was less intra-practitioner variability when using the CQP on the same patient model. NASA TLX revealed that the CQP did increase the cognitive workload (CQP group=16.51%±8.11 vs. 12.22%±8.07 [p=0.251]). The CQP also achieved a high SUS score of 91±7.28. CONCLUSION: The CQP significantly improved the reproducibility and accuracy of placing precordial electrodes V1, V3-V6 with little additional cognitive effort, and with a high degree of usability.

Multipotent adult progenitor cells on an allograft scaffold facilitate the bone repair process.

Multipotent adult progenitor cells are a recently described population of stem cells derived from the bone marrow stroma. Research has demonstrated the potential of multipotent adult progenitor cells for treating ischemic injury and cardiovascular repair; however, understanding of multipotent adult progenitor cells in orthopedic applications remains limited. In this study, we evaluate the osteogenic and angiogenic capacity of multipotent adult progenitor cells, both in vitro and loaded onto demineralized bone matrix in vivo, with comparison to mesenchymal stem cells, as the current standard. When compared to mesenchymal stem cells, multipotent adult progenitor cells exhibited a more robust angiogenic protein release profile in vitro and developed more extensive vasculature within 2 weeks in vivo. The establishment of this vascular network is critical to the ossification process, as it allows nutrient exchange and provides an influx of osteoprogenitor cells to the wound site. In vitro assays confirmed the multipotency of multipotent adult progenitor cells along mesodermal lineages and demonstrated the enhanced expression of alkaline phosphatase and production of calcium-containing mineral deposits by multipotent adult progenitor cells, necessary precursors for osteogenesis. In combination with a demineralized bone matrix scaffold, multipotent adult progenitor cells demonstrated enhanced revascularization and new bone formation in vivo in an orthotopic defect model when compared to mesenchymal stem cells on demineralized bone matrix or demineralized bone matrix-only control groups. The potent combination of angiogenic and osteogenic properties provided by multipotent adult progenitor cells appears to create a synergistic amplification of the bone healing process. Our results indicate that multipotent adult progenitor cells have the potential to better promote tissue regeneration and healing and to be a functional cell source for use in orthopedic applications.

Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development.

UNLABELLED: Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures. SIGNIFICANCE: Human adult marrow stromal stem cells have shown great potential in addressing unmet health care needs. Quality assurance is imperative to make advancements in large-scale manufacturing procedures. MicroRNAs are master regulators of biological processes and potentially ideal quality markers. MicroRNA and mRNA profiling data of two human adult stem cell types were correlated to biological functions in silico. Doing this provided evidence that differentially expressed microRNAs are involved in regulating specific stem cell features. Furthermore, expression of a selected microRNA panel was maintained in next-generation culturing platforms, demonstrating the robustness of microRNA profiling in stem cell comparability testing.

International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an "open-access" manner, such as through publication or database collection.

Current perspectives on the use of ancillary materials for the manufacture of cellular therapies.

Continued growth in the cell therapy industry and commercialization of cell therapies that successfully advance through clinical trials has led to increased awareness around the need for specialized and complex materials utilized in their manufacture. Ancillary materials (AMs) are components or reagents used during the manufacture of cell therapy products but are not intended to be part of the final products. Commonly, there are limitations in the availability of clinical-grade reagents used as AMs. Furthermore, AMs may affect the efficacy of the cell product and subsequent safety of the cell therapy for the patient. As such, AMs must be carefully selected and appropriately qualified during the cell therapy development process. However, the ongoing evolution of cell therapy research, limited number of clinical trials and registered cell therapy products results in the current absence of specific regulations governing the composition, compliance, and qualification of AMs often leads to confusion by suppliers and users in this field. Here we provide an overview and interpretation of the existing global framework surrounding AM use and investigate some common misunderstandings within the industry, with the aim of facilitating the appropriate selection and qualification of AMs. The key message we wish to emphasize is that in order to most effectively mitigate risk around cell therapy development and patient safety, users must work with their suppliers and regulators to qualify each AM to assess source, purity, identity, safety, and suitability in a given application.

Multipotent adult progenitor cells for hypoxic-ischemic injury in the preterm brain.

BACKGROUND: Preterm infants are at risk for hypoxic-ischemic encephalopathy. No therapy exists to treat this brain injury and subsequent long-term sequelae. We have previously shown in a well-established pre-clinical model of global hypoxia-ischemia (HI) that mesenchymal stem cells are a promising candidate for the treatment of hypoxic-ischemic brain injury. In the current study, we investigated the neuroprotective capacity of multipotent adult progenitor cells (MAPC®), which are adherent bone marrow-derived cells of an earlier developmental stage than mesenchymal stem cells and exhibiting more potent anti-inflammatory and regenerative properties. METHODS: Instrumented preterm sheep fetuses were subjected to global hypoxia-ischemia by 25 min of umbilical cord occlusion at a gestational age of 106 (term ~147) days. During a 7-day reperfusion period, vital parameters (e.g., blood pressure and heart rate; baroreceptor reflex) and (amplitude-integrated) electroencephalogram were recorded. At the end of the experiment, the preterm brain was studied by histology. RESULTS: Systemic administration of MAPC therapy reduced the number and duration of seizures and prevented decrease in baroreflex sensitivity after global HI. In addition, MAPC cells prevented HI-induced microglial proliferation in the preterm brain. These anti-inflammatory effects were associated with MAPC-induced prevention of hypomyelination after global HI. Besides attenuation of the cerebral inflammatory response, our findings showed that MAPC cells modulated the peripheral splenic inflammatory response, which has been implicated in the etiology of hypoxic-ischemic injury in the preterm brain. CONCLUSIONS: In a pre-clinical animal model MAPC cell therapy improved the functional and structural outcome of the preterm brain after global HI. Future studies should establish the mechanism and long-term therapeutic effects of neuroprotection established by MAPC cells in the developing preterm brain exposed to HI. Our study may form the basis for future clinical trials, which will evaluate whether MAPC therapy is capable of reducing neurological sequelae in preterm infants with hypoxic-ischemic encephalopathy.

Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury.

Following spinal cord injury (SCI), immune-mediated secondary processes exacerbate the extent of permanent neurological deficits. We investigated the capacity of adult bone marrow-derived stem cells, which exhibit immunomodulatory properties, to alter inflammation and promote recovery following SCI. In vitro, we show that human multipotent adult progenitor cells (MAPCs) have the ability to modulate macrophage activation, and prior exposure to MAPC secreted factors can reduce macrophage-mediated axonal dieback of dystrophic axons. Using a contusion model of SCI, we found that intravenous delivery of MAPCs one day, but not immediately, after SCI significantly improves urinary and locomotor recovery, which was associated with marked spinal cord tissue sparing. Intravenous MAPCs altered the immune response in the spinal cord and periphery, however biodistribution studies revealed that no MAPCs were found in the cord and instead preferentially homed to the spleen. Our results demonstrate that MAPCs exert their primary effects in the periphery and provide strong support for the use of these cells in acute human contusive SCI.

Solution-Phase Crosstalk and Regulatory Interactions Between Multipotent Adult Progenitor Cells and Peripheral Blood Mononuclear Cells.

UNLABELLED: Multipotent adult progenitor cells (MAPCs) are adult adherent stromal stem cells currently being assessed in clinical trials for acute graft versus host disease with demonstrated immunomodulatory capabilities and the potential to ameliorate detrimental autoimmune and inflammation-related processes. Anti-CD3/anti-CD28 (3/28) activation of T cells within the peripheral blood mononuclear cell (PBMC) compartment was performed in the presence or absence of MAPCs. Liquid chromatography-coupled tandem mass spectrometry was used to characterize the differential secretion of proteins, and transcriptional profiling was used to monitor mRNA expression changes in both cell populations. Overall, 239 secreted and/or ectodomain-shed proteins were detected in the secretomes of PBMCs and MAPCs. In addition, 3/28 activation of PBMCs induced differential expression of 2,925 genes, and 22% of these transcripts were differentially expressed on exposure to MAPCs in Transwell. MAPCs exposed to 3/28-activated PBMCs showed differential expression of 1,247 MAPC genes. Crosstalk was demonstrated by reciprocal transcriptional regulation. Secretome proteins and transcriptional signatures were used to predict molecular activities by which MAPCs could dampen local and systemic inflammatory responses. These data support the hypothesis that MAPCs block PBMC proliferation via cell cycle arrest coupled to metabolic stress in the form of tryptophan depletion, resulting in GCN2 kinase activation, downstream signaling, and inhibition of cyclin D1 translation. These data also provide a plausible explanation for the immune privilege reported with administration of donor MAPCs. Although most components of the major histocompatibility complex class II antigen presentation pathway were markedly transcriptionally upregulated, cell surface expression of human leukocyte antigen-DR is minimal on MAPCs exposed to 3/28-activated PBMCs. SIGNIFICANCE: This study documents experiments quantifying solution-phase crosstalk between multipotent adult progenitor cells (MAPCs) and peripheral blood mononuclear cells. The secretome and transcriptional changes quantified suggest mechanisms by which MAPCs are hypothesized to provide both local and systemic immunoregulation of inflammation. The potential impact of these studies includes development of a robust experimental framework to be used for preclinical evaluation of the specific mechanisms by which beneficial effects are obtained after treatment of patients with MAPCs.