Genetically activated mesenchymal stem cells and stromal cells

Abstract

The present disclosure is directed to the generation of modified mesenchymal stem / stromal cells (MSCs) that express IRF1 in increased levels as compared to the starting MSC source, such as MSC cells lines or primary cells. The cells have the advantage of not only improving their longevity in a variety of settings, such also selectively activating expression of certain therapeutic molecules, such as IDO1 and COX-2, while avoiding problematic induction of signaling pathways such as that involving STAT1 and including MHC class II molecules. These cells are therefore uniquely useful in treating autoimmune diseases and disorders.

Description

[0001]DESCRIPTION GENETICALLY ACTIVATED MESENCHYMAL STEM CELLS AND STROMAL CELLS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under grant no. 2041107 awarded by the National Science Foundation and grant no. R21EB030772 awarded by the National Institutes of Health. The government has certain rights in the invention. PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 589,589, filed October 11, 2023, the entire contents of which are hereby incorporated by reference. INCORPORATION OF A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on October 8, 2024, is named RICEP0147WO.xml and is 12,338 bytes in size. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields medicine, immunology, cell biology and genetic engineering. More particularly, the disclosure relates to the engineering of mesenchymal stem / stromal cells to express interferon regulatory factor 1 (IRF1) and the use of such cells to treat autoimmune conditions, as well as to prepare conditioned medium for similar uses. 2. Background Mesenchymal stem / stromal cells (MSCs) are widely recognized for their potential for treating diverse classes of human diseases and disorders (Zhuang et al., 2021). The HI¿FDF\ of MSCs or their cell-free products (Lotfy et al., 2023; Montero-Vilchez et al., 2021) has been studied in hundreds of clinical trials (Levy et al., 2020) across multiple settings spanning 4882-8762-3149, v.1 neurodegenerative disorders (Gugliandolo et al., 2020)^^LQÀDPPDWRU\^ERZHO^GLVHDVH (Eiro et al., 2022), cardiac diseases (Guo et al., 2020), coronavirus disease 2019 (Yao et al., 2022), and others. This broad potential stems from the ability of MSCs to simultaneously express and secrete a spectrum of different pleiotropic immunomodulatory and regenerative chemokines, cytokines, growth factors, metabolites, and vesicles (Pittenger et al., 2019; Han et al., 2022). Virtually all MSC-based cell therapies seek to leverage the delivery of these effector molecules WR^VXSSUHVV^LQÀDPPDWLRQ^^UHVWRUH^LPPXQH^KRPHRVWDVLV^^DQG^SURPRWH^KHDOLQJ (Li et al., 2023; Ma et al., 2014). Despite their promise, MSC therapies have shown limited clinical HI¿FDF\ (Wright et al., 2021). Multiple factors contribute to this GH¿FLHQF\^ many of which can be linked to LQVXI¿FLHQW^ FHOO^ SRWHQF\ (Levy et al., 2020; Giri & Galipeau, 2020). Overall, it is widely accepted that MSCs must be primed biochemically (Kim et al., 2018; Almeida-Porada et al., 2020) or biophysically (Diaz et al., 2017; Xu et al., 2022) to activate and / or enhance their production of immune effector molecules since basal unstimulated MSCs do not typically SURGXFH^WKHVH^PROHFXOHV^LQ^VXI¿FLHQW^TXDQWLWLHV^^3UR-LQÀDPPDWRU\^VWLPXOL including cytokines such as interferon g (IFN-g), tumor necrosis factor a (TNF-Į), and interleukin 1b (IL-1b) or pathogenic molecules such as lipopolysaccharide (Kurte et al., 2020), individually and in combination (Chen et al., 2023), have been explored extensively due to their ability to mimic natural priming of MSCs by activated immune cells during infections or injuries (Noronha et al., 2019). These molecules induce comprehensive changes to the MSC transcriptome, proteome, and secretome (Wiese et al., 2022) via the activation of stimulus-responsive transcription factors (TFs) (Kim et al., 2018). As a key example, IFN-g bolsters MSC potency through the TF signal transducer of activation 1 (STAT1) (Vigo et al., 2017). Stimulation with IFN-g activates STAT1 phosphorylation, dimerization, and translocation to the nucleus, where it binds to gamma-activated sequences within the promoters of many different IFN-stimulated genes (ISGs) (Michalska et al., 2018; Ivashkiv & Donlin, 2014; Ivashkiv, 2018). This includes additional TFs that can function as signaling intermediates, like IFN response factor 1 (IRF1), which further broaden transcriptional responses to IFN-g signaling by binding to IFN- stimulated response elements within the promoters of additional IRGs, notably with and / or without STAT1 (Rosain et al., 2023; Zhang et al., 2015). Among other effectors, IRF1 is well known to regulate indoleamine 2,3-dioxygenase 1 (IDO1) (Schmalzl et al., 2022; 4882-8762-3149, v.1 Campbell et al., 2014), which is recognized as a key determinant of the immunosuppressive potency of MSCs (Chinnadurai et al., 2018; Lim et al., 2021; Ling et al., 2014). Although biochemical and biophysical / biomechanical priming methods have both been shown to boost MSC bioactivity and improve therapeutic responses, several challenges remain that affect the functionality and durability of primed MSCs. In particular, MSC activation tends to be transient and short lived (Zimmermann et al., 2017). The effect of biochemical priming by IFN-g and other stimulants has been shown to decay within a few days in stimulus washout experiments (Gonzalez et al., 2017). More persistent activation has been achieved using engineered biomaterials that integrate the slow release of IFN-g to provide constant stimulation (Zimmermann et al., 2017). Yet, biochemical stimulation is DOVR^GLI¿FXOW^ WR^PRGLI\^DQG^FDQ LQGXFH^ WKH^ H[SUHVVLRQ^ RI^ XQZDQWHG^ DQG^ GHOHWHULRXV^ LPPXQRJHQLF^ DQG^RU^ SURLQÀDPPDWRU\^ effectors (Tse et al., 2003; Stagg et al., 2006). For example, in addition to other SURLQÀDPPDWRU\^ IDFWRUV^^ ,)1-g strongly induces the expression of the transcriptional coactivator class II transcriptional activator (CIITA) (Lee et al., 2008). CIITA, in turn, upregulates the expression of multiple major histocompatibility complex (MHC) class II molecules that can potentiate MSC immunogenicity and promote MSC clearance by CD4 T cells (Alagesan et al., 2018). MSCs have been genetically engineered to constitutively overexpress transgenes encoding for different effector molecules including IDO1 (Xie et al., 2022), COX-2 (Li et al., 2015), and other effectors (Haghighitalab et al., 2021). While constitutive overexpression of these molecules can address the durability of MSC activation, current genetic engineering technologies can typically only manipulate the expression of a few genes at a time, far less than the number of genes that are activated via biochemical or biomechanical stimuli. The multifaceted secretome of MSCs, especially of primed MSCs, facilitates regulation of multiple classes of immune and immune-supporting cells (Song et al., 2020), and this is among the unique properties that make MSCs attractive candidates for cell therapy. 4882-8762-3149, v.1 SUMMARY Thus, in accordance with the present disclosure there is provided a mesenchymal stem cell / mesenchymal stromal cell engineered comprising an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell. The IRF1 expression may be under control of a constitutive promoter, such as any eukaryotic version of the Cytomegalovirus Promoter, Elongation Factor-1 Alpha Promoter, Simian Virus 40 Promoter, Phosphoglycerate Kinase Promoter, EF-^Į^ 3URPRWHr, CMV-HTLV Hybrid Promoter, CMV Early Enhancer / Chicken Beta-Actin Promoter, Glyceraldehyde 3-Phosphate Dehydrogenase Promoter, U6 promoter, or Heat Shock Protein Promoter. The IRF1 expression may be under the control of a conditional / inducible promoter, such as a Tet-inducible promoter, lac-inducible promoter, T7 inducible promoter, IPTG-inducible promoter, or Gal-inducible promoter. The IRF1 may be truncated IRF1 but retains the ability to activate expression of IDO1, COX2, IL4I1, PD- / ^^^F3 / $^Į^^) / *^^^DQG^RU^*DOHFWLQ^^. The IRF1 coding region may be truncated, and only include DNA base pairs 0 through 867 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only includes DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only include 5%, 20%, or 50% of the DNA base pairs 0 to 867 and 100% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only include 100% of the DNA base pairs 0 to 867 and 5%, 20%, or 50% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. Corresponding IRF1 sequences in reference to of SEQ ID NO: 3 are also contemplated. The cell(s) may be encapsulated in a biomaterial such as alginate. The biomaterial may be modified with anti-inflammatory or anti-fibrotic small molecules. The cells may be treated before or after engineering with a biochemical stimulus such as budesonide, dexamethasone, 71)Į^^,)1Ȗ^^ / LSRSRO\VDFFKDULGH^^ / 36^^^, / -^ȕ^^RU^RWKHUV^ The exogenous transcription cassette amy be a non-viral vector or a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral (HSV) vector, a baculoviral vector, a vesicular stomatitis virus (VSV) vector, or others. The IRF1 coding region may be codon optimized. The cell may be a human cell or a non-human mammalian cell. The cell may be further engineered to express a selectable marker, such as an antibiotic resistance gene product or a fluorescent / chemiluminescent gene product. The cell may be further engineered to express a 4882-8762-3149, v.1 suicide gene under control of a conditional / inducible promoter. The cell may be an hTERT- modified cell or a primary adipose-derived cell. The cell may express indoleamine 2,3- dioxygenase 1 (IDO1) at increased levels as compared to a comparable cell not engineered to expression IRF1, such as with 30% or more IDO1-highly expressing cells at 7 days or more following engineering, such as expressing IDO1 at increased levels for at least 1 week, at least two 2 weeks, at least three weeks, at least a month, or at least three months. Also provided is a mesenchymal stem cell / mesenchymal stromal cell treated or engineered to express interferon regulatory factor 1 (IRF1) protein or an mRNA coding for IRF1. The mRNA coding for IRF1 may be codon optimized. The IRF1 may be truncated IRF1 but retains the ability to activate expression of IDO1 and / or COX-2. The cell may be a human cell or a non-human mammalian cell. The cell may be engineered to express a selectable marker, such as an antibiotic resistance gene product or a fluorescent / chemiluminescent gene product. The cell may be engineered to express a suicide gene under control of a conditional / inducible promoter. The cell may be an hTERT-modified cell or a primary MSC derived from adipose, bone marrow, umbilical chord, or from an induced pluripotent cell (iPSC). The cell may express indoleamine 2,3-dioxygenase 1 (IDO1) at increased levels as compared to a comparable cell not engineered to expression IRF1, such as with 30% or more IDO1-highly expressing cells at 7 days or more following engineering, such as wherein the cell expresses IDO1 at increased levels for at least 1 week, at least two 2 weeks, at least three weeks, at least a month, or at least three months. In another aspect, there is provide a method of increasing the in vivo half-life of a mesenchymal stem cell / mesenchymal stromal cell comprising (a) engineering the cell to contain an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell or (b) contacting the cell with interferon regulatory factor 1 (IRF1) or an mRNA encoding IRF1; or a method of increasing the number of passages a mesenchymal stem cell / mesenchymal stromal cell will sustain comprising (a) engineering the cell to contain an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell or (b) contacting the cell with interferon regulatory factor 1 (IRF1) or an mRNA encoding IRF1. In yet another aspect, there is provided a method of producing a conditioned medium comprising culturing the cell as described here in a basal medium. The culturing may occur 4882-8762-3149, v.1 for about 24-72 hours. Also provided is a conditioned medium produced according to such method. In still yet another aspect, there is provided a method of treating an autoimmune disease in a subject comprising administering the cell as described herein to a subject. The autoimmune disease may be atherosclerosis, graft versus host disease, multiple sclerosis, Crohn’s disease, Rheumatoid Arthritis, Type 1 Diabetes, Psoriasis, or Acute Respiratory Distress Syndrome. The method may further comprise administering to said subject a second autoimmune disease therapy, such as an anti-inflammatory agent / immunomodulatory agent. The subject may be a human or non-human mammal. The subject may be administered the cell more than one, such as on a chronic basis, such as weekly, monthly, quarterly, semi-annually, or annually. The non- viral vector may be a transposon vector such as a Sleeping Beauty, a PiggyBac vector, or others. The IRF1 coding region may be truncated, and only include DNA base pairs 0 through 867 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only include DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only include 5%, 20%, or 50% of the DNA base pairs 0 to 867 and 100% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. The IRF1 coding region may be truncated, and only include 100% of the DNA base pairs 0 to 867 and 5%, 20%, or 50% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon. Corresponding IRF1 sequences in reference to of SEQ ID NO: 3 are also contemplated. The cell(s) may be encapsulated in a biomaterial such as alginate. The biomaterial may be modified with anti-inflammatory or anti-fibrotic small molecules. The cells may be treated before or after engineering with a biochemical stimulus such as budesonide, dexamethasone, 71)Į^^,)1Ȗ^^ / LSRSRO\VDFFKDULGH^^ / 36^^^, / -^ȕ^^RU^RWKHUV^ The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of 4882-8762-3149, v.1 illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGS. 1A-E. Genetic priming MSCS via overexpression of IRF1. (FIG. 1A) IRF1 lentiviral construct design. (FIG.1B) Flow-based quantification of lentiviral transduction efficiency. Images were created using BioRender. (FIG. 1C) IRF1 transgene expression and IFN- g stimulation at 50 ng / mL for 24 h both yield nuclear localization of IRF1. IRF1 was visualized by immunocytochemical staining. Scale bar, 100 mm. (FIG. 1D and FIG. 1E) IRF1 overexpression and IFN-g stimulation (50 ng / mL for 24 h) both drive IDO1 transcription (FIG. 1D) and upregulate kynurenine production (FIG.1E). Relative expres- sion, 2DDCt, in RT-qPCR analyses was calculated relative to unprimed MSCs for both MSCIFN-gand MSCIRF1. Statistical analyses were performed using multiple unpaired t tests. Kynurenine production was assayed using an Ehrlich reaction and analyzed using one-way ANOVA. *p < 0.05. Error bars represent the SD. FIGS.2A-G. MSCIRF1recapitulates signatures of MSCIFN-gactivation and T cell suppression. (FIG. 2A) Volcano plot from RNA-seq analyses for MSCIFN-g(left) and MSCIRF1(right). Red and blue regions denote down and up-regulated DEGs respectively (FC > 2; p < 0.05). (FIG. 2B) Expression heatmap for DEGs identified for MSCIFN-g. Genes were ordered from highest to lowest FC. Corresponding FCs for MSCIRF1are provided. (FIG.2C) Venn diagram of all DEGs identified for MSCIFN-gand MSCIRF1. (FIG. 2D) Normalized enrichment scores for GO terms associated with T cells suppression / activation. (FIG. 2E and FIG. 2F) Suppression of T cell activation in flow cytometry based, CellTrace Violet dilution assays. T cells in PBMC cultures were activated using anti-CD3 and anti-CD28 antibodies and cultured with MSC CM. (FIG. 2G) CD4 T cells were found to express less TNF-Į on a per-cell basis with CM from 4882-8762-3149, v.1 MSCIFN-gand MSCIRF1compared with unstimulated MSCs 3 days after exposure to MSC CM. One way ANOVA. *p < 0.05, **p < 0.01. ns, not significant. Error bars represent the SD. FIGS. 3A-E. MSCIRF1avoids MHC class II expression and subsequent CD4 T cell activation and proliferation present with MSCIFN-g. (FIG.3A) Relative expression levels of CIITA and MHC class II genes for MSCIFN-gand MSCIRF1with respect to unprimed MSCs. (FIG. 3B and FIG. 3C) Flow cytometry analyses of HLA-DR surface expression. (FIG. 3D and FIG. 3E) Quantitation of TNF-Į expression in CD4+T cells (FIG. 3D) and PBMC proliferation (FIG. 3E) upon direct co-culture with MSCIFN-gthan MSCIRF1cells. Percent proliferation values were calculated using CellTrace Violet intensities. One-way ANOVA. *p < 0.05, **p < 0.01. ns, not significant. Error bars represent the SD. FIGS. 4A-D. MSCIRF1avoids STAT1 phosphorylation and subsequent signaling present in MSCIFN-gin favor of nuclear localization of unphosphorylated STAT1. (FIG. 4A) Activation of STAT1 transcription by IFN-g stimulation and IRF1 transgene overexpression. (FIG. 4B) Western blotting confirms STAT1 phosphorylation occurs in MSCIFN-g, but not MSCIRF1or unprimed MSCs. (FIG. 4C) Immunocytochemical imaging of STAT1 and pSTAT1 localization. Images are contrasted equivalently. Scale bar, 100 mm. (FIG. 4D) Summary quantification of nuclear localization of pSTAT1. One-way ANOVA. Error bars represent the SD.****p < 0.0001. FIGS.5A-B. MSCIRF1does not undergo morphological changes exhibited byMSCIFN-g. (FIG.5A) Representative fluorescence images of ER (red) and nuclei (Hoechst / blue) in control MSCs, MSCIRF1, and MSCIFN-g. The green lines depict cell edges determined via CellProfiler. Scale bar, 100 mm. (FIG. 5B) Volcano plots of measured changes in morphological features relative to unmodified naive MSC for MSCIFN-g(top) and MSCIRF1(bottom) show significant morphological adaptations occur in MSCIFN-gthat do not occur in MSCIRF1. Dashed lines set to FC of 1.25. FIGS. 6A-F. CM from primary MSCIFN-gand MSCIRF1abrogate T cell proliferation and activation. (FIG. 6A) IDO1 expression measured by flow cytometry shows IRF1 expression results in nearly as much IDO1 expression as IFN-g stimulation. (FIG. 6B) Kynurenine production, an indicator of MSC activity, was high and indistinguishable between MSCIFN-gand MSCIRF1. (FIG. 6C) MSCIFN-gabrogated T cell proliferation slightly more than MSCIRF1. Severe proliferation of more than 3 generations was abrogated by both MSCIFN-gand MSCIRF1. (FIG. 6D) Summary graph of FIG. 6C. (FIG. 6E) MSCIFN-gand MSCIRF1were similar 4882-8762-3149, v.1 in their ability to minimize CD8 T cell activation via IFN-g expression. (FIG. 6F) MSCIFN-gand MSCIRF1were similar in their ability to minimize CD4 T cell activation via TNF-Į expression. One way ANOVA. *p < 0.05, ****p < 0.0001. ns, not significant. Error bars represent the SD. FIGS. 7A-C. Persistence of IRF1 mediated MSC activation. (FIG. 7A) Experimental scheme for MSC transduction and transient treatment with IFN-g and / or budesonide. (FIG. 7B) IDO1 expression responses in non-engineered MSCs that were primed biochemically with 50 ng / mL IFN-g and / or with 1 mg / mL budesonide (BUD) for 24 h (FIG. 7C) The effect of IFN-g and / or budesonide on the activation and persistence of IRF1- transduced MSCs. Percent positive IDO1 cells were determined by flow cytometry. Error bars represent the SD, n = 3. FIGS. S1A-D. IRF1 Lentivirus Titering. Transduction efficiency was analyzed via flow cytometry in (FIG. S1A) immortalized human adipose MSCs and (FIG. S1B) primary human adipose MSCs that were transduced with variable amounts of IRF1 lentivirus. (FIG. S1C) Exemplar gating strategy of GFP positive MSCs used to assess MOI in A and B. (FIG. S1D) GFP and brightfield images of three replicates of IRF1-transduce immortalized adipose 06&V^^ / HQWLYLUXV^FRQWDLQHG^ERWK^,5)^^DQG^H*)3^XQGHU^VHSDUDWH^SURPRWHUV^^6FDOH^EDU^^^^^P^ FIGS. S2A-B. MSCIRF1avoids T cell activation while largely maintaining ability to negatively regulate T cell proliferation. GSEA analysis of RNAseq for (FIG. S2A) MSCIFN-gand (FIG. S2B) MSCIRF1for GO terms associated with T cell activation and proliferation. FIGS. S3A-D. Flow cytometry gating schemes for assessing MSC immunomodulation of cultured PBMCs. (FIGS. S3A-D) PBMC viability, T cell activation, and T cell proliferation were quantified via flow cytometry using the gating scheme presented in each panel. Representative data for PBMCs at day 3 (FIGS. S3A-B), and day 6 (FIGS. S3C- D) are shown. PBMCs were activated with anti- CD3 and anti-CD28 antibodies day zero. Untr = PBMCs not treated with MSC conditioned media. FIGS. S4A-D. Original images of western blots for FIG. 4C of the main text. Western blot analysis of (FIG. S4A) STAT1, (FIG. S4B) pSTAT1, (FIG. S4C) IRF1, and (FIG. S4D) ȕ-Tubulin in hTERT-MSCs 8 days after IRF1 transduction. FIGS. S5A-D. STAT1 phosphorylation is temporarily present in MSCIRF1shortly after lentiviral transduction, but then returns to low baseline Western blot of hTERT-MSCs characterized (FIG. S5A) 4 days following IRF1 transduction showing activation of STAT1 phosphorylation due to IRF1 lentivirus exposure. White arrows denote 4882-8762-3149, v.1 nuclear position. The corresponding western blot in FIG. 4C of the main text shows this activation fades by day 8, indicating it stems from lentivirus exposure. (FIG. S5B) Immunofluorescence images of hTERT-MSCs show that a control GFP lentivirus does not LQGXFH^,5)^^H[SUHVVLRQ^RU^QXFOHDU^ORFDOL]DWLRQ^^6FDOH^EDU^^^^^P^^(FIG. S5C-D) Entire original western blot images for panel A of (FIG. S5C) IRF1 (top) and pSTAT1 (bottom) and (FIG. S5D) 67$7^^^OHIW^^DQG^ȕ-Tubulin (left). FIG. S6. MSCIRF1does not undergo morphological changes experienced by MSCIFN-g. Distribution of morphological metrics from microscopy analyses of features that changed most significantly across MSC groups: nuclear Area / ER area ratio, ER form factor, ER compactness, ER convex area, ER perimeter, ER major axis length, ER max feret diameter. FIG. S7. Gating and results of IRF1 lentiviral tittering of primary MSCs. Flow cytometry analysis of primary human adipose MSCs transduced with varied amounts of IRF1 lentivirus. FIGS. S8A-C. RNAseq of primary adipose MSCs reveals IRF1 engineering maintains therapeutic signature, MSC identity, and paracrine function effector Rap1. (FIG. S8A). Volcano plots labeled with negative regulators of T cell activation and proliferation. (FIG. S8B) Expressed and non-expressed markers of MSC identity were largely maintained in all conditions, albeit with exception of HLA-DR in AD-MSC,)1Ȗ. (FIG. S8C) Rap1 expression, a marker whose deficiency can impair paracrine function of MSCs, is maintained. FIGS. S9A-C. MSCIRF1cells maintain high viability but show reduced proliferation (FIG. S9A) Representative low cytometry analyses of primary adipose MSCs stained with viability dyes Propidium Iodide and Annexin V. (FIG. S9B) Summary of panel A. (FIG. S9C) Growth curve with MSC,)1Ȗunder FRQVWDQW^ ,)1Ȗ^ VWLPXODWLRQ^ TXDQWLILHG^ E\^ WU\SDQ^ EOXH^ staining and counting. FIGS. S10A-C. Flow cytometry gating schemes for PBMCs cultured with conditioned media from primary human adipose-derived MSCs Representative measurements of PBMC viability and immunophenotype are shown for day 3 (FIG. S10A). Viability and T cell proliferation were quantified at day 7 (FIGS. S10B-C). FIGS. S11A-C. IDO1 activation by IRF1 overexpression in different commercial AD- MSC batches, after AD-MSC freezing and thawing, and in iPSC derived MSCs. (FIG. S11A) IRF1 engineering yielded variable levels of IDO1 expression 7 days after transduction of primary adipose MSCs depending on patient source and freeze / thaw cycle. (FIGS. S11B-C) 4882-8762-3149, v.1 Flow cytometry histograms show IDO1 levels in IRF1-transduced iPSC- derived MSCs were higher at day 21 compared to day 7, yielded near equivalent percent cell activation compared to primary adipose MSCs (FIG. S11C). FIG. S12. MHC Class II molecule HLA-DR expression in IRF1 modified cells with and without ,)1Ȗ and budesonide. Flow cytometry analyses were performed 10 days IROORZLQJ^,5)^^WUDQVGXFWLRQ^^,)1Ȗ^^EXGHVRQLGH^^%8'^^RU^ERWK^ZHUH^DSSOLHG^^^^KRXUV^EHIRUH^ measurements. FIG. S13. Kynurenine production from NTC hTERT-MSCs (control), IRF1 CRISPRa hTERT-MSCs, and IRF1 transgene hTERT-MSCs. FIGS. S14A-D. LPS is another biochemical stimulant that may boost IDO1 expression following IRF1 engineering. (FIG. S14A) RNAseq shows the addition of LPS to IRF1 engineered hTERT-MSCs resulted in heightened expression of IDO1 and other anti- inflammatory genes. (FIG. S14B) LPS-responsive anti-inflammatory genes were not only the result of LPS alone. (FIG. S14C) Principal component analysis shows the combination of IRF1 and LPS results in a transcriptional program that is very different from either IRF1 or LPS alone. (FIG. S14D) LPS achieves transcriptional responses without STAT1 phosphorylation. FIGS. S15A-C. Pro-inflammatory MHC class II expression, seen in both immortalized and primary MSCs LQ^UHVSRQVH^WR^,)1Ȗ, is not exacerbated by LPS. (FIG. S15A) RNAseq indicates MHC class II expression is activated by IFNȖ in both primary and immortalized human adipose MSCs. (FIG. S15B) RNAseq indicates MHC class II molecule expression seen with MSCIFNȖis not exacerbated by LPS at the RNA level in hTERT-MSCs. (FIG. S15C) Exemplary staining and flow cytometry of HLA-DR protein further supports LPS does not exacerbate MHC class II expression in hTERT-MSCs. 4882-8762-3149, v.1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Here, the inventors demonstrate that stable hyperexpression of IRF1 can be leveraged to upregulate broad arrays of immunomodulatory effector genes and recapitulate the potency gains that can be achieved via traditional biochemical priming with IFN-g. However, genetic priming via IRF1 was also shown to circumvent the activation of STAT1 and key STAT1- dependent downstream ISGs like CIITA. In contrast with IFN-g-primed cells, this effect was associated with low MHC class II gene expression and improved retention of MSC hypo- immunogenicity in MSC-peripheral blood mononuclear cell (PBMC) coculture assays. Finally, sustained (>21-day) priming was also demonstrated in primary human adipose-derived MSCs (AD-MSCs), where activated IDO1 production was maintained for weeks. Together, these results demonstrate that the engineered expression of intermediate TFs like IRF1 can facilitate selective and persistent programming of MSCs in ways that activate large numbers of therapeutically relevant effector molecules while minimizing the activation of other molecules that have the potential to LPSDLU^WKHUDSHXWLF^HI¿FDF\^ Such control may open routes to better tailor and maintain the DFWLYDWHG^SKHQRW\SHV^RI^06&V^IRU^VSHFL¿F^WKHUDSHXWLF^DSSOLFDWLRQV^ These and other aspects of the disclosure are described in detail below. I. Mesenchymal Stem / Stromal Cells Mesenchymal stem cells (MSCs), also known as mesenchymal stromal cells, medicinal signaling cells, or multipotent stromal cells, can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue). While the terms mesenchymal stem cell (MSC) and marrow stromal cell have been used interchangeably for many years, neither term is sufficiently descriptive. Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. While this is an accurate description for one function of MSCs, the term fails to convey the relatively recently discovered roles of MSCs in the repair of tissue. The term encompasses multipotent cells derived from other non- marrow tissues, such as placenta, umbilical cord blood, adipose tissue, adult muscle, corneal 4882-8762-3149, v.1 stroma, or the dental pulp of deciduous (baby) teeth. The cells do not have the capacity to reconstitute an entire organ. Mesenchymal stem cells (MSCs) are characterized morphologically by a small cell body with a few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. The cells, which are long and thin, are widely dispersed, and the adjacent extracellular matrix is populated by a few reticular fibrils, but is devoid of the other types of collagen fibrils. These distinctive morphological features of mesenchymal stem cells can be visualized label-free using live cell imaging. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define MSCs. A cell can be classified as an MSC if it shows plastic adherent properties under normal culture conditions and has a fibroblast-like morphology. In fact, some argue that MSCs and fibroblasts are functionally identical. Furthermore, MSCs can undergo osteogenic, adipogenic and chondrogenic differentiation ex vivo. The cultured MSCs also express on their surface CD73, CD90 and CD105, while lacking the expression of CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR surface markers. MSCs are found throughout the human body. Bone marrow was the original source of MSCs, and is still the most frequently utilized source. These bone marrow stem cells do not contribute to the formation of blood cells, and so do not express the hematopoietic stem cell marker CD34. They are sometimes referred to as bone marrow stromal stem cells. The youngest and most primitive MSCs may be obtained from umbilical cord tissue, namely Wharton's jelly and umbilical cord blood. However, MSCs are found in much higher concentration in the Wharton's jelly compared to cord blood, which is a rich source of hematopoietic stem cells. The umbilical cord is available after birth. It is normally discarded, and poses no risk for collection. These MSCs may prove to be a useful source of MSCs for clinical applications, due to their primitive properties and fast growth rate. These cells have several advantages over bone-marrow-derived MSCs. Adipose-tissue- derived MSCs (AdMSCs), in addition to being easier and safer to isolate than bone-marrow- derived MSCs, can be obtained in larger quantities. 4882-8762-3149, v.1 Adipose-tissue-derived MSCs (AdMSCs), in addition to being easier and safer to isolate than bone-marrow-derived MSCs, can be obtained in larger quantities. The developing tooth bud of the mandibular third molar is a rich source of MSCs. While they are described as multipotent, it is possible that they are pluripotent. They eventually form enamel, dentin, blood vessels, dental pulp, and nervous tissues. These stem cells are capable of differentiating into chondrocytes, cardiomyocytes, melanocytes, and KHSDWRF\WH^OLNH^ FHOOV in vitro. Stem cells are present in amniotic fluid. As many as 1 in 100 cells collected during amniocentesis are pluripotent mesenchymal stem cells. MSCs have a great capacity for self-renewal while maintaining their multipotency. 5HFHQW^ ZRUN^ VXJJHVWV^ WKDW^ ȕ-catenin, via regulation of EZH2, is a central molecule in maintaining the "stemness" of MSC's. The standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes and chondrocytes as well as myocytes. MSCs have been seen to even differentiate into neuron-like cells, but doubt remains about whether the MSC-derived neurons are functional. The degree to which the culture will differentiate varies among individuals and how differentiation is induced, e.g., chemical vs. mechanical; and it is not clear whether this variation is due to a different amount of "true" progenitor cells in the culture or variable differentiation capacities of individuals' progenitors. The capacity of cells to proliferate and differentiate is known to decrease with the age of the donor, as well as the time in culture. Likewise, whether this is due to a decrease in the number of MSCs or a change to the existing MSCs is not known. MSCs have an effect on innate and specific immune cells, and research has shown an ability to suppress tumor growth. MSCs produce many immunomodulatory molecules including prostaglandin E2 (PGE2), nitric oxide, indoleamine 2,3-dioxygenase (IDO), interleukin 6 (IL-6), and other surface markers such as FasL, PD-L1 and PD-L2. MSCs have an effect on macrophages, neutrophils, NK cells, mast cells and dendritic cells in innate immunity. MSCs are able to migrate to the site of injury, where they polarize through PGE2 macrophages in M2 phenotype which is characterized by an anti-inflammatory effect. Further, PGE2 inhibits the ability of mast cells to degranulate and produce TNF- Į^ Proliferation and cytotoxic activity of NK cells is inhibited by PGE2 and IDO. MSCs also reduce the expression of NK cell receptors - NKG2D, NKp44 and NKp30. MSCs inhibit respiratory flare and apoptosis of neutrophils by production of cytokines IL-6 and IL- 4882-8762-3149, v.1 8. Differentiation and expression of dendritic cell surface markers is inhibited by IL-6 and PGE2 of MSCs. The immunosuppressive effects of MSC also depend on IL-10, but it is not certain whether they produce it alone, or only stimulate other cells to produce it. MSC expresses the adhesion molecules VCAM-1 and ICAM-1, which allow T- lymphocytes to adhere to their surface. Then MSC can affect them by molecules which have a short half-life and their effect is in the immediate vicinity of the cell. These include nitric oxide, PGE2, HGF, and activation of receptor PD-1. MSCs reduce T cell proliferation between G0 and G1 cell cycle phases DQG^GHFUHDVH^WKH^H[SUHVVLRQ^RI^,)1Ȗ^RI^7K^^FHOOV^ZKLOH^LQFUHDVLQJ^ the expression of IL-4 of Th2 cells. MSCs also inhibit the proliferation of B-lymphocytes between G0 and G1 cell cycle phases. MSCs produce several antimicrobial peptides (AMPs) including human cathelicidin LL-37, ȕ-defensins, lipocalin 2 and hepcidin. These peptides, together with the enzyme indoleamine 2,3-dioxygenase (IDO), are responsible for the broad-spectrum antibacterial activity of MSCs. Mesenchymal stem cells can be activated and mobilized in reaction to injury and infection. As of May 2023, ClinicalTrials.gov lists more than 1,100 studies featuring MSCs for more than 920 conditions. Clinical studies investigating the efficacy of mesenchymal stem cells in treating diseases are in clinical development around the world, particularly treating autoimmune diseases, graft versus host disease, Crohn's disease, multiple sclerosis, systemic lupus erythematosus and systemic sclerosis. Many of the early clinical successes using intravenous transplantation came in systemic diseases such as graft versus host disease and sepsis. Direct injection or placement of cells into a site in need of repair may be the preferred method of treatment, as vascular delivery suffers from a "pulmonary first pass effect" where intravenous injected cells are sequestered in the lungs. Further studies into the mechanisms of MSC action may provide avenues for increasing their capacity for tissue repair. The majority of modern culture techniques still take a colony-forming unit-fibroblasts (CFU-F) approach, where raw unpurified bone marrow or ficoll-purified bone marrow mononuclear cells are plated directly into cell culture plates or flasks. Mesenchymal stem cells, but not red blood cells or hematopoietic progenitors, are adherent to tissue culture plastic within 24 to 48 hours. However, at least one publication has identified a population of non-adherent MSCs that are not obtained by the direct-plating technique. 4882-8762-3149, v.1 Other flow cytometry-based methods allow the sorting of bone marrow cells for specific surface markers, such as STRO-1. STRO-1+ cells are generally more homogenous and have higher rates of adherence and higher rates of proliferation, but the exact differences between STRO-1+ cells and MSCs are not clear. Methods of immunodepletion using such techniques as MACS have also been used in the negative selection of MSCs. The supplementation of basal media with fetal bovine serum or human platelet lysate is common in MSC culture. Prior to the use of platelet lysates for MSC culture, the pathogen inactivation process is recommended to prevent pathogen transmission. II. Interferon Regulatory Factor 1 (IRF1) Interferon regulatory factor 1 (IRF1) is a protein that in humans is encoded by the IRF1 gene. IRF1 was the first member of the interferon regulatory transcription factor (IRF) family identified. Initially described as a transcription factor able to activate expression of the cytokine ,)1ȕ, IRF-1 was subsequently shown to function as a transcriptional activator or repressor of a variety of target genes. IRF-1 regulates expression of target genes by binding to an interferon stimulated response element (ISRE) in their promoters. The IRF-1 protein binds to the ISRE via an N-terminal helix-turn-helix DNA binding domain, which is highly conserved among all IRF proteins. Beyond its function as a transcription factor, IRF-1 has also been shown to trans- activate the tumor suppressor protein p53 through the recruitment of its co-factor p300. IRF-1 has been shown to play roles in the immune response, regulating apoptosis, DNA damage and tumor suppression. It has been shown that the extreme C-terminus of IRF-1 regulates its ability to activate transcription, nanobodies targeting this domain (MF1) are able to increase IRF-1 activity. IRF1 has been shown to interact with CHIP, GAGE, HSP70 / HSP90, IRF8, KPNA2, MYD88, PCAF, STAT1, TAT, VEGFR2, and REDD2. Representative sequences for mouse and human IRF1, DNA and protein are set forth in SEQ ID NOS: 1-4. SEQ ID NO: 1 – human IRF1 DNA sequence: Atgcccatcactcggatgcgcatgagaccctggctagagatgcagattaattccaaccaaatcccggggctcatctggattaataaag aggagatgatcttccagatcccatggaagcatgctgccaagcatggctgggacatcaacaaggatgcctgtttgttccggagctgggc cattcacacaggccgatacaaagcaggggaaaaggagccagatcccaagacgtggaaggccaactttcgctgtgccatgaactccc 4882-8762-3149, v.1 tgccagatatcgaggaggtgaaagaccagagcaggaacaagggcagctcagctgtgcgagtgtaccggatgcttccacctctcacc aagaaccagagaaaagaaagaaagtcgaagtccagccgagatgctaagagcaaggccaagaggaagtcatgtggggattccagc cctgataccttctctgatggactcagcagctccactctgcctgatgaccacagcagctacacagttccaggctacatgcaggacttgga ggtggagcaggccctgactccagcactgtcgccatgtgctgtcagcagcactctccccgactggcacatcccagtggaagttgtgcc ggacagcaccagtgatctgtacaacttccaggtgtcacccatgccctccacctctgaagctacaacagatgaggatgaggaagggaa attacctgaggacatcatgaagctcttggagcagtcggagtggcagccaacaaacgtggatgggaaggggtacctactcaatgaacc tggagtccagcccacctctgtctatggagactttagctgtaaggaggagccagaaattgacagcccagggggggatattgggctgagt ctacagcgtgtcttcacagatctgaagaacatggatgccacctggctggacagcctgctgaccccagtccggttgccctccatccagg ccattccctgtgcaccgtag SEQ ID NO: 2 – mouse IRF1 DNA sequence: Atgccaatcactcgaatgcggatgagaccctggctagagatgcagattaattccaaccaaatcccagggctgatctggatcaataaag aagagatgatcttccagattccatggaagcacgctgctaagcacggctgggacatcaacaaggatgcctgtctgttccggagctgggc cattcacacaggccgatacaaagcaggagaaaaagagccagatcccaagacatggaaggcaaacttccgttgtgccatgaactccct gccagacatcgaggaagtgaaggatcagagtaggaacaagggcagctctgctgtgcgggtgtaccggatgctgccacccctcacca ggaaccagaggaaagagagaaagtccaagtccagccgagacactaagagcaaaaccaagaggaagctgtgtggagatgttagcc cggacactttctctgatggactcagcagctctaccctacctgatgaccacagcagttacaccactcagggctacctgggtcaggacttg gatatggaaagggacataactccagcactgtcaccgtgtgtcgtcagcagcagtctctctgagtggcatatgcagatggacattatacc agatagcaccactgatctgtataacctacaggtgtcacccatgccttccacctccgaagccgcaacagacgaggatgaggaagggaa gatagccgaagaccttatgaagctctttgaacagtctgagtggcagccgacacacatcgatggcaagggatacttgctcaatgagcca gggacccagctctcttctgtctatggagacttcagctgcaaagaggaaccagagattgacagccctcgaggggacattgggataggc atacaacatgtcttcacggagatgaagaatatggactccatcatgtggatggacagcctgctgggcaactctgtgaggctgccgccctc tattcaggccattccttgtgcaccatag SEQ ID NO: 3 – human IRF1 protein amino acid sequence: MPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRS WAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRML PPLTKNQRKERKSKSSRDAKSKAKRKSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGY MQDLEVEQALTPALSPCAVSSTLPDWHIPVEVVPDSTSDLYNFQVSPMPSTSEATTDE DEEGKLPEDIMKLLEQSEWQPTNVDGKGYLLNEPGVQPTSVYGDFSCKEEPEIDSPG GDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPSIQAIPCAP* SEQ ID NO: 4 – mouse IRF1 protein amino acid sequence: 4882-8762-3149, v.1 MPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRS WAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRML PPLTRNQRKERKSKSSRDTKSKTKRKLCGDVSPDTFSDGLSSSTLPDDHSSYTTQGYL GQDLDMERDITPALSPCVVSSSLSEWHMQMDIIPDSTTDLYNLQVSPMPSTSEAATD EDEEGKIAEDLMKLFEQSEWQPTHIDGKGYLLNEPGTQLSSVYGDFSCKEEPEIDSPR GDIGIGIQHVFTEMKNMDSIMWMDSLLGNSVRLPPSIQAIPCAP* III. Engineering of Cells In certain embodiments, cells are transformed or engineered with expression cassettes to express IRF1. Provided herein are such expression cassette and expression vectors which contain one or more nucleic acids encoding IRF1. Additional elements such as selectable / detectable markers and control sequences may also be introduced into cells. Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers / promoters from both viral and eukaryotic sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection / detection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide. A. Regulatory Elements Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one 4882-8762-3149, v.1 or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. 4882-8762-3149, v.1 Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Below is a list of promoters / enhancers and inducible promoters / enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter / enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. The promoter and / or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and / or DQ E, E-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, E-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, D-fetoprotein, t-globin, E-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), D1-antitrypain, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia virus. In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), E-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, D-2- macroglobulin, vimentin, MHC class I gene H-2Nb, HSP70, proliferin, tumor necrosis factor, 4882-8762-3149, v.1 and / or thyroid stimulating hormone D gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), ElA, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, serum, interferon, SV40 large T antigen, PMA, and / or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein. Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. B. Delivery of Expression Vectors There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells. Retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5’ and 3’ ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a 4882-8762-3149, v.1 recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells. A particular type of retrovirus is lentivirus. The virus contains a reverse transcriptase molecule found to perform transcription of the viral genetic material upon entering the cell. Within the viral genome are RNA sequences that code for specific proteins that facilitate the incorporation of the viral sequences into the host cell genome. The "gag" gene codes for the structural components of the viral nucleocapsid proteins: the matrix (MA / p17), the capsid (CA / p24) and the nucleocapsid (NC / p7) proteins. The "pol" domain codes for the reverse transcriptase and integrase enzymes. Lastly, the "env" domain of the viral genome encodes for the glycoproteins and envelope on the surface of the virus. There are multiple steps involved in the infection and replication of a lentivirus in a host cell. In the first step the virus uses its surface glycoproteins for attachment to the outer surface of a cell. More specifically, lentiviruses attach to the CD4 glycoproteins on the surface of a host's target cell. The viral material is then injected into the host cell's cytoplasm. Within the cytoplasm, the viral reverse transcriptase enzyme performs reverse transcription of the viral RNA genome to create a viral DNA genome. The viral DNA is then sent into the nucleus of the host cell where it is incorporated into the host cell's genome with the help of the viral enzyme integrase. From now on, the host cell starts to transcribe the entire viral RNA and express the structural viral proteins, in particular those that form the viral capsid and the envelope. The lentiviral RNA and the viral proteins then assemble and the newly formed virions leave the host cell when enough are made. One method of gene therapy involves modifying a virus to act as a vector to insert beneficial genes into cells. Unlike other retroviruses, which cannot penetrate the nuclear envelope and can therefore only act on cells while they are undergoing mitosis, lentiviruses can infect cells whether or not they are dividing. Many cell types, like neurons, do not divide in adult organisms, so lentiviral gene therapy is a good candidate for treating conditions that affect those cell types. 4882-8762-3149, v.1 Some experimental applications of lentiviral vectors have been done in gene therapy in order to cure diseases like diabetes mellitus, murine hemophilia A, prostate cancer, chronic granulomatous disease, and vascular diseases. Therapy requires manipulation of the lentivirus genes and structure for delivery of specific genes to alter the course of the disease. Parts of the viral genome must be removed so that the virus can't replicate itself. It is replaced with a gene to permanently incorporate into the host cell's genome using genetically modified virus. HIV-derived lentiviral vectors have been used for introducing libraries of complementary DNAs, short hairpin RNAs, and cis-regulatory elements into many targets, including embryonic stem cells. Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as adenovirus, vaccinia virus, adeno-associated virus (AAV), and herpes viruses can also be used. Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use. Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. 4882-8762-3149, v.1 This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product. In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000TMis widely used and commercially available. In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) to facilitate fusion with the cell membrane and promote cell entry of liposome- encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present 4882-8762-3149, v.1 disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase. Other expression constructs which can be employed to deliver nucleic acids encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific. Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells. IV. Pharmaceutical Formulations and Methods of Treatment A. Formulation and Administration The present disclosure provides pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol 4882-8762-3149, v.1 monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Generally, the ingredients of compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. B. Treating Autoimmune Disease As discussed herein, the presently claimed cells, conditioned media and cell preparations may be used to treating autoimmune disease and disorders. The include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, 4882-8762-3149, v.1 autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, systemic scleroderma, progressive systemic sclerosis (PSS), systemic sclerosis (SS), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus (SLE), Takayasu arteritis, temporal arteritis / giant cell arteritis, inflammatory bowel disease (IBD), ulcerative colitis, Cohn's disease, intestinal mucosal inflammation, wasting disease associated with colitis, uveitis, vitiligo and Wegener's granulomatosis, Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy, ventilator induced lung injury, viral infections, autoimmune diabetes and the like. Inflammatory disorders include, for example, chronic and acute inflammatory disorders. It may also be desirable to provide combination treatments using cells, cell preparations and media of the present disclosure in conjunction with additional autoimmune / anti- inflammatory therapies. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameters. This process may involve contacting the subject with both agents / therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell / subject with two distinct compositions or formulations, at the same time, wherein one composition includes 4882-8762-3149, v.1 the antibody and the other includes the other agent. The other agent could be steroids or non- steroidal anti-inflammatory or other immune modulating (e.g., immunosuppressive) substance. Alternatively, the cells, cell preparations and media of the present disclosure may precede or follow the other treatment by intervals ranging from minutes to weeks. One would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapies would still be able to exert an advantageously combined effect on the cell / subject. In such instances, it is contemplated that one would contact the subject with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the interim time period for treatment significantly, such as to avoid toxicity, including where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Various combinations may be employed. For the example below the cells, cell preparations and media of the present disclosure is “A” and the second autoimmune therapy is “B”: A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A Administration of any cell or therapy of the present disclosure to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. V. Examples The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many 4882-8762-3149, v.1 changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 - Materials and Methods MSC culture. Immortalized human adipose hTERT-MSCs, primary AD-MSCs, and iPSC-derived MSCs were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). hTERT-MSCs were maintained between passage 2 and 15 in DMEM (Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Corning, Corning, NY, USA) and 1% antibiotic-antimycotic (Gibco, Carlsbad, CA, USA). For AD-MSCs, genetic priming effects were characterized in separate batches of MSCs from ATCC from two different lots. For iPSC-derived MSCs, one lot from ATCC was used. AD-MSCs and iPSC-derived MSCs were maintained between passages 2 and 8 in Mesenchymal Stem Cell Basal Medium for Adipose, Umbilical, and Bone Marrow-derived MSCs (PCS-500-030, ATCC) with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs - Low Serum (PCS-500-040, ATCC) Passaging was done using 0.25% trypsin for 5 min at 37oC. Primary MSCs were thawed immediately from the manufacturer and used in downstream experiments XQOHVV^RWKHUZLVH^VSHFL¿HG^^,Q^IUHH]H-thaw experiments, primary MSCs were frozen at passage 3 in FBS with 10% DMSO before being thawed. MSCs were treated with human recombinant IFN-g (PeproTech, Cranbury, NJ, USA) at a concentration of 50 ng / mL for 24 hr ZKHUH^LQGLFDWHG^XQOHVV^RWKHUZLVH^VSHFL¿HG^ Lentiviral MSC engineering. The human IRF1 viral vector was synthesized by VectorBuilder (Chicago, IL, USA). HEK293T cells were obtained from ATCC and maintained in the same media as the hTERT-MSCs between passage 2 and 30. The HEK cells were plated at 7.7E4 cells / cm2the day before transfection. For each transfection, 4.878 mg IRF1 lentiviral plasmid, 1.463 mg pMD2.G, and 3.659 mg psPAX2 (Addgene, Watertown, MA, USA) were transfected using JetPRIME (Polyplus, Illkirch, France). Media was exchanged for media containing 4 mM sodium butyrate after 8 h. Virus-containing media was harvested at 24 and 48 hr, ¿OWHUHG^ ZLWK^ D^ ^^^^-PP^ ¿OWHU^ WR^ UHPRYH^ FHOOXODU^ GHEULV^ DQG^ concentrated (100x) using Lenti-X concentrator (Takara, San Jose, CA, USA). The resulting particles were resuspended in DMEM with 10% FBS, 1% antibiotic-antimycotic and stored at -80oC. 4882-8762-3149, v.1 The IRF1 lentivirus was reverse transduced into immortalized or primary MSCs in a six- well plate. MSCs between passages 2 and 5 were added onto the lentivirus and 10 mM polybrene was added to each well. Cells were then incubated for 72 hr, their media were changed, and they were incubated for another 3–72 hr before their GFP production was assessed E\^ÀRZ^F\WRPHWU\^WR^GHWHUPLQH^WUDQVGXFWLRQ^HI¿FLHQF\^ An MOI of 1 was the minimum viral volume to obtain approximately 100% GFP positive cells. This viral volume (MOI = 1) was used in replicate reverse transduction process to make biological replicates of transgenic IRF1 MSCs that were used in downstream H[SHULPHQWV^^7UDQVGXFWLRQ^HI¿FLHQFLHV^ZHUH^FRQ¿UPHG^ YLD^ÀXRUHVFHQFH^LPDJLQJ^ RT-qPCR. Immortalized human adipose MSCs were plated in 6 well plates and rested for 24 hr, after which, cells were lysed, and RNA was extracted using a RNeasy Mini Kit (Qiagen, Germantown, MD, USA). RNA was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). qPCR was conducted using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and the following primers (IDT, Newark, NJ, USA): IRF1 F: 5’-CTCTCCCCGACTGGCACATC-3’(SEQ ID NO: 5) IRF1 R: 5’-CCGACTGCTCCAAGAGCTTCA-3’(SEQ ID NO: 6) IDO1 F: 5’-ACGGGACACTTTGCTAAAGGC-3’(SEQ ID NO: 7) IDO1 R: 5’-GGTTGCCTTTCCAGCCAGACA-3’(SEQ ID NO: 8) GAPDH F: 5’-CAATGACCCCTTCATTGACC-3’(SEQ ID NO: 9) GAPDH R: 5’-TTGATTTTGGAGGGATCTCG-3’(SEQ ID NO: 10) RNA-seq. RNA-seq experiments were performed in duplicate. RNA was extracted using an RNEasy Mini Kit and frozen at -20oC and sent to Azenta (Burlington, MA, USA) for whole RNA-VHT^^)$674^¿OHV^ZHUH^SURFHVVHG^XVLQJ^*DOD[\^WR^TXDQWLI\^UHDG^FRXQWV^IRU^HDFK^ gene and condition. R and Python were used to convert Ensemble gene IDs to gene symbols. Python and the GSEAPY package were used to conduct GO enrichment analyses. Western blotting. MSCs were lysed in RNA immunoprecipitation buffer. Total protein concentrations were TXDQWL¿HG using a BCA assay. The inventors loaded 25 mg protein for SDS-PAGE and transferred onto a PVDF membrane for western blot. Primary antibodies for STAT1 (66545-1, Proteintech, Rosemont, IL, USA) and pSTAT1 (MA5-15071, ThermoFisher 6FLHQWL¿F^ Waltham, MA, USA) were used at a 1:5,000 and 1:1,000 dilutions, respectively, in 1x Tris-buffered saline with 1% casein (Bio- Rad). Secondary a-mouse 4882-8762-3149, v.1 horseradish peroxidase (HRP) (A6154, Sigma-Aldrich, St. Louis, MO, USA) or a-rabbit HRP (ab6721, Ab- cam, Cambridge, UK) were used at a 1:3,000 dilution in 1x Tris-buffered saline with 1% casein. Membranes were exposed after addition of ECL (170-5060, Bio-Rad). Tubulin was detected with hFAB Rhoda- mine Anti-Tubulin Primary Antibody (12004166, Bio-Rad) at 1:3,000 dilution. Immunocytochemistry. MSCs were plated in six-well plates containing coverslips, cultured for 24 hr, and ¿xed with 4% PFA in PBS for 20 min at 4oC–8oC. Cells were washed thoroughly with 0.2-mm ¿ltered 5% BSA in PBS (blocking solution), and permeabilized by incubation in 0.2% Triton X-100 in PBS at room temperature for 5 min. After washing and incubating with blocking solution for 1 hr, cells were immuno-stained overnight at 4oC–8oC with rabbit anti-human IRF1 antibody (1:1,000, ab243895, Abcam) or rabbit anti-human pSTAT1 (1:400, MA5- ^^^^^^^7KHUPR)LVKHU^6FLHQWL¿F^^DQG^PRXVH^DQWL-human STAT1 (1:800, 66545-1, Proteintech) diluted in blocking buffer. Secondary antibody staining was performed using Cy5 Goat anti-UDEELW^ ^^^^^^^^$^^^^^^^7KHUPR)LVKHU^6FLHQWL¿F^^^$)^^^^^GRQNH\^DQWL- rabbit (1:500, A21207, Invitrogen, Waltham, MA, USA), or AF 647 goat anti-mouse (1:500, A21235, LTC, Carlsbad, CA, USA). Secondary staining solutions also contained Hoechst nuclear stain (1:10,000, 33342, ThermoFisher 6FLHQWL¿F^^ Cells were visualized with a Nikon Eclipse Ti2 at 60x PDJQL¿FDWLRQ^ Background subtraction was done using ImageJ and was SHUIRUPHG^LGHQWLFDOO\^IRU^HDFK^ÀXRURSKRUH^ Flow cytometry. &HOOV^ ZHUH^ WU\SVLQL]HG^^ ¿[HG^ XVLQJ^ %'^ &\WRSHUP^&\WR¿[^ ^%'^^ Franklin Lakes, NJ, USA), permeabilized with BD Perm / Wash Buffer, and stained at room temperature for 1 hr using a PE-labeled mouse anti-IDO1 antibody (12-9477-42, ThermoFisher 6FLHQWL¿F^^RU^D^%9^^^-labeled mouse anti-human HLA-DR antibody (564041, BD) according to the manufacturer’s instructions. Excess antibody was removed via centrifugation and VXSHUQDWDQW^UHPRYDO^^)OXRUHVFHQFH^OHYHOV^ZHUH^WKHQ^TXDQWL¿HG^ Kynurenine production. Kynurenine concentration was assessed by Ehrlich reaction. The inventors combined 100 mL CM with 50 mL 30 wt% trichloroacetic acid in water in a round-bottom 96-well plate. A standard curve of SXUL¿HG L-kynurenine (Sigma-Aldrich) dissolved in R10 media was also included. This mixture was spun down at 2200RCF. They combined 100 mL supernatant with fresh 2 wt% dimethylaminobenzaldehyde (Sigma-Aldrich) LQ^JODFLDO^DFHWLF^DFLG^^7KHUPR)LVKHU^6FLHQWL¿F^^^7KLV^PL[WXUH^ZDV^LPPHGLDWHO\^UHDG^DW^^^^^QP^ on a plate reader. 4882-8762-3149, v.1 Suppression bioactivity assays. MSC CM was prepared by culturing primary MSCs (2 x 105cells) or hTERT-MSCs (1 x 106cells) in R10 media for 48–72 hr (RPMI Medium 1640 (Gibco, Waltham, MA, USA) with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Gibco, Grand Island, NY), 1% GlutaMAX Supplement (Gibco, Grand Island, NY), 1% sodium pyruvate 100 mM (Gibco, Grand Island, NY), 1% HEPES 1 M (Gibco, Grand Island, NY), 1% MEM non-essential amino acids (Gibco, Grand Island, NY) for 48 and 72 hr, respectively. CM was used directly without storage in subsequent assays. Human PBMCs were prepared from buffy coats obtained from Gulf Coast Regional Blood Center (Houston, TX, USA). Buffy coats were frozen and stored in liquid nitrogen before being thawed just before use. Viability was measured by AOPI (Nexcelom, Lawrence, MA, USA) and was required to be greater than 90% at thaw. T cells were activated by incubating PBMCs with anti-CD3 (OKT3, Tonbo / Cytek, San Diego, CA, USA) and anti-CD28 (302934, Bio- legend, San Diego, CA, USA) antibodies in PBS at 37oC for 3–5 h. For T cell proliferation analyses, PBMCs were stained with CellTrace 9LROHW^^7KHUPR^)LVKHU^6FLHQWL¿F^^EHIRUH^DFWLYDWLRQ^ TNF-Į and IFN-g expression levels were measured by treating PBMCs with GolgiPlug ^7KHUPR^ )LVKHU^ 6FLHQWL¿F^^ DQG^ *ROJL6WRS^ ^7KHUPR^ )LVKHU^ 6FLHQWL¿F^^ WR^ IDFLOLWDWH^ LPPXQRVWDLQLQJ^DQDO\VHV^^3%0&V^ZHUH^¿[HG^DQG^SHUPHDELOL]HG^IRU^^ hr at room temperature with Foxp3 / TF Fix / Perm Concentrate and Diluent (Tonbo / Cytek), and then washed with Tonbo Flow Cytometry Perm Buffer (Tonbo / Cytek). Cells were then immuno-stained 1 hr at room temperature with a panel of antibodies: anti-CD3 (563725, BD), anti-TNF-Į (Biolegend 502938), anti-IFN-Į (Biolegend 506516), anti-perforin (Biolegend 506516), anti-T-bet (Biolegend 644812), and anti-Ki67 (BD 564071) for. Viable cells were LGHQWL¿HG using Ghost Dye Violet 510 (Tonbo / Cytek). Flow cytometry was performed using a BD LSRFortessa LQVWUXPHQW^^7^FHOO^SUROLIHUDWLRQ^DQG^DFWLYDWLRQ^ZHUH^TXDQWL¿HG^LGHQWLFDOO\^LQ^06&-PBMC co- culture experiments. In these experiments, MSCs were cultured for 3 days before PBMCs were placed in coculture for an additional 7 days. Media were changed every other day. Immortalized human adipose MSCs were plated at 5.2E3 cells / cm2in 6 well plates. Cells were then stained with ER-Tracker Red ^7KHUPR^ )LVKHU^ 6FLHQWL¿F^ (^^^^^^^ DQG^ +RHFKVW^ QXFOHDU^G\H^^7KHUPR^)LVKHU^6FLHQWL¿F^^^^^^^^DFFRUGLQJ^WR^PDQXIDFWXUHU^SURWRFROV^^&HOOV^ZHUH^ visualized using a Nikon Eclipse Ti2. A background subtraction method with a rolling ball radius and a contrast enhancement was done to better visualize cells in ImageJ. &HOO3UR¿OHU was 4882-8762-3149, v.1 used to identify cellular components. Nuclei and ER were LGHQWL¿HG by applying a size ¿lter, separating clumped objects, and ¿lling holes within objects. CellProlifer module MeasureObjectSizeShape was then used to mea- sure morphological features of nuclei and ER. Subsequent data processing was done using MATLAB. Example 2 – Results IRF1 overexpression mimics key activation signatures of IFN-g priming. The usefulness of IRF1 transgenes for priming was ¿rst tested using immortalized hTERT-MSCs that were transduced (multiplicity of infection [MOI] = 1) with a lentiviral vector that encoded for a full length IRF1 transgene and eGFP (FIG.1A, FIG. 1B, and FIGS. S1A-D). The resulting IRF1-overexpressing cells (MSCIRF1) displayed enhanced IRF1 transcription, translation, and nuclear localization by RT-qPCR and immunocytochemistry (FIGS. 1C and 1D). Although biochemical stimulation with IFN-g for 24 hr (MSCIFN-g) induced higher transcription of endogenous IRF1 compared with MSCIRF1, both methods yielded a more than 25,000-fold induction of IDO1 transcription (FIG. 1D), which is a key ISG target of IRF1 and a signature of IFN-g priming. IDO1 exerts immunomodulatory effects by converting tryptophan into kynurenine within the tryptophan metabolic pathway (Stone & Williams, 2023). Kynurenine levels were also elevated using both priming techniques and were indistinguishable between MSCIRF1and MSCIFN-g(p = 0.63) (FIG. 1E). Together these results demonstrate that IRF1 transgene expression in MSCs can recapitulate key activation signatures of IFN-g stimulation. IRF1 activates MSC-mediated T cell suppression. The inventors next characterized and compared IRF1 and IFN-g-induced changes to the MSC transcriptome using bulk RNA sequencing (RNA-seq) (FIGS. 2A–2D). Both priming methods induced transcriptional changes, yielding more than 1900 differentially expressed genes (DEGs), each relative to untreated control MSCs (p < 0.05) (fold change [FC] > 2) (FIGS.2A–2C). In addition to IDO1, upregulated DEGs included COX-2 (PTGS2), a second key immunomodulatory effector enzyme that produces the anti-LQÀDPPDWRU\ small molecule prostaglandin E2 (Thumkeo et al., 2022), along with multiple effector genes associated with T cell suppression including IL4I1 (Sadik et al., 2020), Galactin-9 (Zhao et al., 2022; Kim et al., 2015) (LGALS9), PD- L2 (Hurrell et al., 2022) (PDCD1LG2), and FGL2 (Zhao et al., 2023) (FIG. 2A). Along these lines, normalized enrichment scores from gene ontology (GO) analysis show transcription in both MSCIRF1and MSCIFN-gis positively enriched for genes corresponding with 4882-8762-3149, v.1 the GO terms “negative regulation of T cell proliferation” (0042130) and “negative regulation of T cell activation” (005086800) (FIG. 2D and FIGS. S2A-B). Consistent with their transcriptional SUR¿OHV^ IRF1- and IFN-g-mediated priming were both demonstrated to enhance the immunosuppressive bioactivities of MSCs compared with basal, unprimed cells (FIGS. 2E–2G and FIGS. S3A-D). MSC conditioned media (CM) prepared using MSCIRF1and MSCIFN-gwere shown to suppress the proliferation of PBMCs that were pre-stimulated with anti-CD3 and anti-CD28 antibodies to selectively activate T cells ( FIG. 2E and FIG. 2F). Finally, CM from MSCsIRF1was also found to decrease the percentage of CD4 T cells that positively expressed the activation marker TNF-Į relative to CM from basal, unprimed MSCs ( FIG.2G). As with the cell proliferation analyses, similar suppression of CD4 T cells was observed with MSCIRF1and MSCIFN-g. IRF1 hyperexpression maintains the immune evasive status of MSCs by circumventing STAT1 activation. RNA-seq also revealed marked differences between genetic and biochemical priming in the MSCIRF1and MSCIFN-gcells ( FIGS. 2B–2D and FIG. 3A). In particular, genes from the GO term “T cell activation” (0042110) were depleted with MSCIRF1, but positively enriched with MSCIFN-g( FIG. 2D). One of the most upregulated MSCIFN-ggenes in this GO term was CD74, or human leukocyte antigen (HLA) class II histocompatibility antigen g chain CD74, involved in the assembly and WUDI¿FNLQJ^RI^0+& class II complexes (Schröder, 2016). The expression of the transcriptional regulator CIITA, which DJDLQ^ GULYHV^ WKH^ GRZQVWUHDP^ H[SUHVVLRQ^ RI^ 0+&^ FODVV^ ,,^ JHQHV^^ ZDV^ DOVR^ VLJQL¿FDQWO\ upregulated in MSCIFN-g(FC = 126 ± 27) (p = 0.0002) but was much less so in MSCIRF1(FC = 6 ± 1.4) (p = 0.008) (FIG. A). In turn, while nearly all MHC class II genes were upregulated by IFN-g treatment, HLA gene expression was largely unchanged by IRF1 overexpression (FIG. 3A). Consistent with this behavior, Àow cytometry analyses of the MHC class II cell surface receptor HLA-DR FRQ¿UPHG^WKLV^UHVXOW^^ FIG. 3B and FIG. 3C). Here, while the level of HLA- '5^ZDV^HOHYDWHG^VOLJKWO\^^FRPSDUHG^ZLWK^QDLYH^^XQPRGL¿HG^06&V^ the geometric mean of HLA-DR expression in MSCIFN-gwas more than 35x that of MSCIRF1. To test if the above MHC class II expression changes would result in changes in immune reactivity, MSCs, MSCsIFNg, and MSCsIRF1were next co-cultured in direct contact with unstimulated PBMCs. After 7 days, CD4+T cells showed an increase in activation status with MSCIFN-gcompared with MSC or MSCIRF1( FIG.3D). There were 60% more TNF-Į + CD4 T cells with MSCIFN-gcompared with MSCIRF1and 70% more with MSCIFN-gcompared with 4882-8762-3149, v.1 untreated MSC. The PBMCs also proliferated 5.5x more with MSCIFN-gcompared with MSCIRF1and 2.4x more with MSCIFN-gcompared with untreated MSC (FIG. 3E). IRF1 priming circumvents STAT1 activation. To gain mechanistic insight into the differences between genetic and biochemical priming, the inventors next examined levels of STAT1 transcription, phosphorylation, and nuclear translocation in naive MSCs, MSCIRF1, and MSCIFN-g( FIGS. 4A-D). IFN-g was found to induce a much larger change in STAT1 transcription (FC = 11 ± 0.35) (p = 0.0005) compared with IRF1 overexpression (FC = 3.0 ± 0.39) (p = 0.02) ( FIG.4A). IFN-J^ZDV^DOVR^IRXQG^WR^LQGXFH^VLJQL¿FDQW^67$7^^SKRVSKRU\ODWLRQ^ by western blot ( FIG. 4B and FIGS. S4A-D) as expected (Kim et al., 2018). In contrast, IRF1 overexpression resulted in much less STAT1 phosphorylation in MSCs that were cultured for at least 8 days after transduction. STAT1 activation is notably observed on day 4 in western blotting experiments ( FIG. S5A, FIG. S5C, and FIG. S5D), which the inventors attribute to stimulation by the lentivirus. However, this signal dissipates by day 8 ( FIG.4B and FIG. S5B), indicating that IRF1 overexpression does not sustain STAT1 activation on its own. IFN-g also induced much more prominent pSTAT1 nuclear localization in immunocytochemical imaging experiments ( FIG. 4C and FIG. 4D). Together, these results suggest that the IRGs that are differentially expressed in MSCIRF1cells relative to naive MSCs are indeed predominantly activated by IRF1 expression and not STAT1. They further suggest that differences between MSCIRF1and MSCIFN-gtranscriptional SUR¿OHV stem from the ability of IRF1 overexpression to bypass STAT1 phosphorylation and avoid upregulation of STAT1- responsive ISGs that are not activated by IRF1 alone, such as CIITA. To further investigate functional consequences of the different activation modes in MSCIRF1and MSCIFN-gcells, high throughput imaging analyses were performed to quantitatively compare differences in primed cell morphologies ( FIGS. 5A-B). Prior reports have shown that IFN-g can induce morphological changes that coincide with enhancement in immunosuppressive capacity (Klinker et al., 2017). The inventors performed similar analyses using ER tracker dye to stain the MSC endoplasmic reticulum (ER), which extends throughout the cytoplasm, in addition to nuclear staining ( FIGS. 5A-B and FIG. S6). Seven MSC morphological features were found to be VLJQL¿FDQWO\ changed upon IFN-g stimulation in this study. Six of these were replicated from previously documented morphological changes in the literature, and one of these was not previously studied. 6LJQL¿FDQWO\ altered features included increases in ER compactness, convex area, perimeter, major axis length, and max feret diameter 4882-8762-3149, v.1 and decreases in ER form factor and nuclear to ER area ratio. MSCIRF1morphology remained statistically similar to basal MSCs. IRF1 can potentiate primary human MSCs activation persistently. The inventors next examined the usefulness of IRF1 overexpression to sustainably activate the immunosuppressive properties of primary AD-MSCs (FIG. 6A-B and FIGS. S7-12). Here, IRF1 overexpression was again observed to induce IDO1 transcription (FIG. S8A) and translation (FIG. 6A) and boost MSC production of kynurenine ( FIG. 6B). RNA-seq showed that IRF1-PRGL¿HG AD-MSCs also maintained transcriptional SUR¿OHV that are required by the International Society for Cellular Therapy to designate cells as MSCs (FIG. S8B). Moreover, RAP1 (TERF2IP) transcription, which has been reported to be necessary to retain MSC paracrine bioactivity (Zhang et al., 2015; Ding et al., 2018), was also unaffected and remained positive with IRF1 overexpression (FIGS. S8&^^^)LQDOO\^^ÀRZ^F\WRPHWU\^DQDO\VHV^VKRZ IRF1- PRGL¿HG primary AD-MSCs retain high levels of viability (>97%) (FIG. S9A and FIG. S9B), although, as with chronic IFN-g exposure in this experiment, IRF1-mediated genetic priming seems to decrease the proliferation rates (FIG. S9C). CM from IRF1-primed AD-MSCs was also capable of suppressing T cell proliferation in CellTrace dilution assays (FIG. 6C, FIG. 6D, and FIGS. S10A-C). This suppression was accompanied by reduced activation of CD8+and CD4+7^FHOOV^^VLJQL¿HG^E\^D^ORVV^RI^H[SUHVVLRQ^ of IFN-g and TNF-Į, respectively (FIG. 6E and FIG. 6F). Importantly, the CM in these experiments were also notably prepared using 5-fold fewer cells compared with the hTERT- MSC media, indicating that primary cells are not only amenable to IRF1-medated priming, but higher potencies can be achieved. A second sample of commercially sourced primary AD-MSCs notably exhibited lower activation with respect to IDO1 expression (FIGS. S11A). IRF1-mediated priming responses were also attenuated appreciably in primary MSCs that were frozen and banked over short timescales (<7 days) (FIG. S11A), indicating IRF1 activation can vary by MSC preparation and patient source. Nevertheless, IDO1 expression remained highly responsive to IFN-g stimulation in these cells. The inventors thus next tested whether IRF1-mediated genetic priming responses could be improved via brief stimulation with IFN-g and / or budesonide, a glucocorticoid that is well known to potentiate MSC responses to IFN-g (Ankrum et al., 2014) (FIG.7A). The inventors’ banked primary MSCs were transduced with IRF1 lentivirus, treated with IFN-g and / or budesonide for 24 hr, and then monitored by Àow cytometry for IDO1 4882-8762-3149, v.1 expression for 21 days. In contrast with biochemical priming alone (FIG.7B), all combinations of IFN-g and / or budesonide with IRF1 overexpression were capable of yielding persistent IDO1 expression for at least 21 days (FIG. 7C). Either IFN-g or budesonide was found to increase the rates of IDO1 expression at early time points, indicating the potentiation of IRF1 transcriptional activity. The inventors notably also demonstrated persistent, 21-day activation, can be achieved using iPSC-derived MSCs [FIG. S11B and FIG. S11C). Finally, while all treatments that included IFN-g were found to upregulate the expression of HLA-DR, this was not the case with budesonide alone. Instead, the combination of IRF1 priming with budesonide was able to maintain the low levels of HLA-DR expression observed with basal, unprimed MSCs. FIG. S13 shows kynurenine expression comparing IRF1 transgene to CRISPRa. FIGS. S14A-D show that LPS added to IRF1 causes a significant change in transcription in part via an increase in STAT1 transcription, but not phosphorylation. It also shows that one can obtain large increases in transcription of the labeled, therapeutic genes. This is important as it shows additional molecular stimulation of IRF1 cells to get induced activation of therapeutic genes. FIGS. S15A-C show that LPS stimulation in addition to IRF1 overexpression does not cause further upregulation of counteractive, pro-inflammatory HLA class II molecules, which is present with IFN-g stimulation. Example 3 - Discussion MSCs remain an attractive tool for cell therapy due to their unique abilities to secrete arrays of pleotropic immunomodulatory and restorative biomolecules. MSCs, however, require some form of biochemical or biophysical priming to activate the production of these molecules since they are not expressed appreciably without stimulation. Priming via pro-LQÀDPPDWRU\ cytokines such as IFN-g to mimic the reciprocal interactions of MSCs with activated immune cells have been shown to induce broad changes to the MSC transcriptome and secretomes, and improve MSC potency (Kim et al., 2018; Almeida-Porada et al., 2020; Chen et al., 2023). Nevertheless, as demonstrated here and by others, IFN-g-induced potency enhancements are very short lived and only last a few days. This work demonstrates that the constitutive overexpression of the stimulus-responsive TF IRF1 can persistently activate MSC expression of many IFN-g-responsive anti- LQÀDPPDWRU\^JHQHV^^LQFOXGLQJ^NH\^LPPXQRPRGXODWRU\^JHQHV^OLNH^IDO1 and PTGS2 (COX-2), 4882-8762-3149, v.1 as well as other negative regulators of T cell activation. Consistent ZLWK^ WKLV^SUR¿OH^^ ,5)^^ overexpression MSCs were capable of suppressing T cell proliferation and activation in both hTERT-PRGL¿HG^DQG^SULPDU\^KXPDQ^06&V^^\LHOGLQJ^QHDU^HTXLYDOHQW^SRWHQF\^IRXQG^ZLWK^,)1- g priming. IRF1-mediated priming was also found to circumvent the expression of the transcriptional activator CIITA, which is upregulated by IFN-g and drives the expression of MHC class II genes known to potentiate MSC immunogenicity and clearance. The inventors attribute this distinction to the role STAT1 plays in activating CIITA transcription, and the usefulness of IRF1 overexpression to circumvent the phosphorylation of STAT1 in the JAK / STAT pathway. While the relative roles of STAT1 and IRF1 in the upregulation of CIITA are found to vary depending on cell type (Reith et al., 2005), the substantial activation of CIITA and downstream MHC class II genes by IFN-g suggests STAT1 dominates this activation in MSCs. The overexpression of IRF1 was not found to LQÀXHQFH^STAT1 phosphorylation or nuclear localization appreciably via immunocytochemistry or western blotting analyses. Transient IRF1 expression has been reported to promote STAT1 activation in HEK cells (Zenke et al., 2018). However, the inventors’ data do not support such effects in MSCs. Instead, the exogenously expressed IRF1 in the genetically primed MSCIRF1cells seems to operate largely independent of the canonical STAT / JAK pathway, enabling selective activation of IRF1- responsive genes without upregulating pSTAT1. The inventors also demonstrated IRF1-mediated genetic priming can boost the immunomodulatory bioactivities of primary human MSCs. Here, the potency of CM from primary MSCIRF1FHOOV^SURYHG^PRUH^SRWHQW^FRPSDUHG^ZLWK^&0^IURP^K7(57^PRGL¿HG^06&IRF1, with approximately 5-fold fewer cells needed to achieve suppression effects equivalent to those with transient IFN-g stimulation. Nevertheless, the inventors also observed variability in IRF1- mediated priming responses across commercial MSC preparations, donor samples, and after freeze-thaw cycles. Responses were measured with respect to IDO1 expression, a key lentiviral transduction, IRF1-mediated induction of IDO1 seems to be times at times delayed in these cells. In contrast, the onset of IDO1 expression in primary MSCs was notably both rapidly and highly responsive to IFN-g priming in all experiments, despite its subsequent decay. Building on the hypothesis that the delayed MSC activation response stemmed from potential epigenetic effects that restrict IRF1 access to IRG loci in the absence of complete JAK / STAT signaling, the inventors GHPRQVWUDWHG^HI¿FLHQW^DQG^SHUVLVWHQW^06&^DFWLYDWLRQ^FDQ^ 4882-8762-3149, v.1 be achieved by combining IRF1-mediated genetic priming and brief stimulation with IFN-g and / or pharmacological treatment using the corticosteroid budesonide. Both these transient treatments proved capable of accelerating MSC activation (IDO1 expression) appreciably. Moreover, constitutive IFR1 transgene expression was then able to take over and sustain IDO1 activation for at least 21 days, which is 4-fold longer than IFN-g or IFN-g plus budesonide. This persistent activation was also demonstrated using iPCS-derived MSCs, which are recognized for their potential off-the-shelf use in therapy and a resultant ability to circumvent issues surrounding MSC source and patient variability. Finally, while IFN-g stimulation was naturally found to upregulate HLA-DR expression, the combination of budesonide and IRF1 overexpression minimized this activation, suggesting that this combination still circumvents activation of STAT1-responsive ISGs. MSC treatments with budesonide and other glucocorticoids have been shown previously to enhance IFN-g-induced IDO expression across multiple donors and in over- passaged cells (Ankrum et al., 2014). In these studies, activation improvements were notably maximized with continuous exposure to budesonide. This study shows that constitutive IRF1 expression can sustain this effect without additional pharmacological treatments. While the results with AD-MSCs and iPSC-MSCs are promising, additional studies are still needed to both optimize the usefulness of this approach within and across different MSC sources and validate the extent to which persistent genetic priming can boost MSCs HI¿FDF\ in vivo. The attenuated proliferation due to IRF1 overexpression may be limiting and necessitate additional cell engineering. However, the inventors expect this behavior to be much less problematic to approaches that incorporate MSCs within biomaterial hydrogels in order to contain and localize MSCs over the course of therapy (Ghanta et al., 2020; Shi et al., 2023; Huang et al., 2022). These strategies naturally stand to EHQH¿W from persistently bolstered MSCs activation. Overall, they anticipate priming via manipulation of IRF1 and / or other stimulus-responsive TFs, individually and in combination, will ultimately facilitate tailored programming of the immunomodulatory and regenerative properties of the secretomes and associated modes of DFWLRQ^RI^06&V^IRU^VSHFL¿F^FHOO^WKHUDS\^applications. Example 4 – Primary MSC Priming and Longevity The first time that IRF-engineered primary adipose MSCs were transduced with IRF1 lentivirus, the cells began making high quantities of IDO1 days after lentiviral transduction. It 4882-8762-3149, v.1 was expected that a constitutively expressed IRF1 would result in IDO1 expression for an extended period of time. Meanwhile, in literature, the effects of IFNȖ stimulation have been shown to rapidly disappear once IFNȖ stimulation stopped. This may be the reason why MSCs have generally lacked in potency, resulting in poor clinical outcomes. Herein, the inventors hoped to conduct a time course experiment that measured the expression of the therapeutic molecule IDO1 following IRF1 engineering and IFNȖ stimulation. Conditions Round 1 (Patient B, fresh): xMSCIFNȖxMSCIRF1Conditions Round 2 (Patient A, freeze / thaw): xMSCIRF1x MSCIRF1+IFNȖ xMSCIRF1+BUD+IFNȖMethods and Materials: xPrimary adipose MSCso Patient B, freshly thawed from ATCC oPatient A, thaw / freeze / thaw after ATCC receiptx Primary MSC media (ATCC 50-189-670FP and 50-189-671FP)x 5HFRPELQDQW^KXPDQ^,)1Ȗ^^3HSURWHFK^^^^-02)x Budesonidex Polybrene transfection reagent (EMD TR-1003-G)x 0.25% Trypsin (Thermo 25200056)xAnti Anti (Thermo 15240096)x hTERT media / trypsin stop solution (Gibco 10-566-024, 10% FBS, 1% Anti Anti)x BD Cytofix / Cytoperm Kit (BD AB_2869008)x PBSx PE Mouse Anti-Human IDO1 (Invitrogen 12-9477-42)x BV786 Mouse Anti-Human HLA-DR (BD 564041)Primary adipose MSCs were transduced with IRF1 lentivirus at an MOI of approximately 1. 4ʅL of IRF1 lentivirus was added to 15 x 12 wells. 70k MSCs at passage 4 4882-8762-3149, v.1 were added to each well in a total volume of 1mL media per well.0.5ʅL polybrene transfection reagent was added per well. Cells were incubated for 3 days before media was changed and cells were stained with anti-IDO1 and run through a Sony MA900 FACS system. Two days prior to this, 18x 12 wells were plated with 70k MSCs. One day later, 50ng / mL recombinant human IFNȖ was added to 15 wells. IDO1 staining was done by trypsinizing 3 wells per condition (control cells only on day 0), stopping trypsinization with hTERT-MSC media, and spinning cells down at 400g for 5min. Cells were then resuspended in 300ʅL BD Cytofix / Cytoperm solution for 20min at 4 ºC.300ʅL BD Perm / Wash solution was added, and the cells were spun down. Supernatant was removed, and the cells were again resuspended in BD Perm / Wash solution. Cells were then resuspended in 5ʅL anti-IDO1 antibody and 45ʅL BD Perm / Wash per sample. Cells were incubated for 45min at room temperature. Cells were then flowed through a Sony MA900 FACS system. This was repeated on day 3, 7, 10, and 21. Cells were passaged as necessary. The same FACS parameters were used each day. Guy Bedford ran the system on day 21 and swapped the naming of the samples on the FACS system; this is corrected in the data analysis. Results. A surprisingly low expression of IDO1 was yielded from MSCIRF1, and an expected result was obtained from MSC,)1Ȗ. This IRF1 data was much lower than the results previously achieved and noted above. The main difference between this experiment and previous experiments was that these MSCs were from a different ATCC order and a different patient – “Patient B” here and “Patient A” in a first go-round. The inventors did not initially return to Patient A because this cell line had either been frozen down in 5% DMSO in FBS or carried past passage 10 – farther than is recommended. They were concerned that a freeze / thaw would interfere with the effects of IRF1. However, after this result, the inventors thought that maybe the IRF1 response was patient dependent, and a freeze / thaw cycle would have a minimal impact. For this reason, they repeated the experiment with MSCs from Patient A that had been UHIUR]HQ^ DQG^ WKHQ^ WKDZHG^^ 7KH^ LQYHQWRUV^ DOVR^ LQFOXGHG^ VWLPXODQWV^ ,)1Ȗ^ DQG^ FRUWLFRVWHURLG^ budesonide to try to get the IRF1 response started in a short period of time. The inventors hypothesized that perhaps IRF1 could maintain chromatin surrounding the IDO1 locus in an ‘on’ state (think DNA and histone methylation and acetylation, open vs closed chromatin, etc.), but IRF1 may not be good at initiating the switching from the ‘off’ to the ‘on’ state. Budesonide has been noted in literature (Ankrum et al., Sci Rep 4, 4645, 2014,) to improve MSCs IDO1 4882-8762-3149, v.1 H[SUHVVLRQ^LQ^UHVSRQVH^WR^,)1Ȗ^DQG^KDV^EHHQ^SUHYLRXVO\^LQYHVWLJDWHG^E\^WKH^inventors, including determining the concentration to be used here. Thawed Patient A primary adipose MSCs were transduced with IRF1 lentivirus the same as above, but in a total of 24 wells. After 3 days, MSC media was changed. 50ng / mL recombinant IFNȖ^ZDV^DGGHG^WR^^^,5)^^ZHOOV^^^ʅg / mL corticosteroid budesonide (BUD) was DGGHG^WR^^^,5)^^ZHOOV^^DQG^ERWK^^^QJ^P / ^,)1Ȗ^DQG^^ʅg / mL budesonide was added to 6 wells. 24 hours later, media was changed on all samples and 3 biological replicates per condition were trypsinized, split, and half of each sample was stained with anti-IDO1 as above, while the other half of each sample was replated for future time points. This was repeated, alternating between available wells on days 3, 7, 10, and 21. Discussion. Temporary stimulation with IFNȖ and / or budesonide following IRF1 transduction seems to supercharge the IRF1 response. This treatment appears to solve the problem of IRF1 / IDO1 reproducibility across patients and freeze / thaw cycles. Future study may be needed to verify this result. * * * * * * * * * * * * * * * * * All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. 4882-8762-3149, v.1 VI. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Alagesan, S., Sanz-Nogués, C., Chen, X., Creane, M., Ritter, T., Ceredig, R., O’Brien, T., and *ULI¿Q^^0^'^^^^^^^^^^$QWL-donor antibody induction following intramuscular injections of allogeneic mesenchymal stromal cells. Immunol. Cell Biol.96, 536–548. Almeida-Porada et al. (2020) Mol. Ther. Methods Clin. Dev. 16, 204–224. Ankrum et al. (2014). Sci. Rep. 4, 4645. Campbell et al. (2014). Front. Neurosci. 8, 12–22. Chen et al., (2023). World J. Stem Cell. 15, 787–806. Chinnadurai et al. (2018) Cell Rep. 22, 2504–2517. Diaz et al. (2017). Biomechanical forces pro- mote immune regulatory function of bone marrow mesenchymal stromal cells. Stem Cell. 35, 1259–1272. Ding et al. (2018). Cell Death Dis. 9, 386. Eiro et al. (2022). Int. J. Mol. Sci. 23. Ghanta et al. (2020). Biomater. Sci. 8, 5061–5070. Giri & Galipeau (2020). Blood Adv. 4, 1987–1997. Gonzalez et al. (2017) Cytotherapy 18, 1114–1128. Gugliandolo et al. (2020). Mesenchymal Stem Cells in Ms.pdf, pp. 1–35. Guo et al. (2020). Cell Death Dis. 11, 349. Haghighitalab et al. (2021). Sci. Rep. 11, 1–19. Han et al. (2022). Signal Transduct. Targeted Ther. 7, 92. Huang et al. (2022). Bioeng. Biotechnol. 10, 1–9. Hurrell et al. (2022). Nat. Commun. 13, 5118. Ivashkiv, L.B. (2018). Nat. Rev. Immunol. 18, 545–558. Ivashkiv, L.B., and Donlin, L.T. (2014). Nat. Rev. Immunol. 14, 36–49. Kim et al. (2015). Immune Netw. 15, 241–251. 4882-8762-3149, v.1 Kim et al. (2018). EBioMedicine 28, 261–273. Klinker et al. (2017). Proc. Natl. Acad. Sci. USA 114, E2598–E2607. / doi.org / 10.1073 / pnas.1617933114. Kurte et al. (2020). Stem Cell Res. Ther. 11, 416. Lee et al. (2008). Eur. J. Immunol. 38, 2325–2336. Levy et al. (2020). Sci. Adv. 6, eaba6884. Li et al. (2015). Int. J. Mol. Med. 35, 1309–1316. Li et al. (2023). Cell. Mol. Immunol. 20, 558–569. Lim et al. (2021). Stem Cell Res. Ther. 12, 37. Ling et al. (2014). Cancer Res. 74, 1576–1587. Lotfy et al. (2023). Stem Cell Res. Ther. 14, 66. Ma et al. (2014). Cell Death Differ. 21, 216–225. Michalska et al. (2018). Front. Immunol. 9, 1135. Montero-Vilchez et al. (2021). Front. Cell Dev. Biol. 9, 654210. Noronha et al. (2019). Cell Res. Ther. 10, 1–21. Pittenger et al. (2019). npj Regen. Med. 4, 22. Reith et al. (2005). Nat. Rev. Immunol. 5, 793–806. Rosain et al. (2023). Cell 186, 621–645.e33. Sadik et al. (2020). Cell 182, 1252–1270.e34. Schmalzl et al. (2022). Nat. Commun. 13, 5730. Schröder, B. (2016). Biochim. Biophys. Acta 1863, 1269–1281. Shi et al. (2023). Acta Pharm. Sin. B 13, 2778–2794. Song et al. (2020). Pharmacol. Sci. 41, 653–664. Stagg et al. (2006). Blood 107, 2570–2577. Stone, T.W., and Williams, R.O. (2023). Cancers 15, 2895. Thumkeo et al. (2022). Cell Rep. 39, 110914. Tse et al. (2003). Transplantation 75, 389–397. 4882-8762-3149, v.1 Vigo et al. T. (2017). J. Allergy Clin. Immunol. 139, 1667–1676. Wiese et al. (2022). Front. Immunol. 13, 917790. Wright et al. (2021). Front. Cell Dev. Biol. 9, 632717. Xie et al. (2022). Stem Cell Res. Ther. 13, 367. Xu et al. (2022). Int. J. Mol. Sci. 23, 4333. Yao et al. (2022). Stem Cell Res. Ther. 13, 124. Zenke et al. (2018). Immunol. Cell Biol. 96, 1095–1103. Zhang et al. (2015). Cell Death Dis. 1, 15007. Zhuang et al. (2021). J. Biomed. Sci. 28, 28. Zhao et al. (2022). Front. Cell Dev. Biol.10, 700702–700717. Zhao et al. (2023). Nature Communications 14, 735. / doi.org / 10.1038 / s41467-023-36430-2. Zimmermann et al. (2017). Stem Cells Transl. Med. 6, 223–237. 4882-8762-3149, v.1

Claims

WHAT IS CLAIMED IS:

1. A mesenchymal stem cell / mesenchymal stromal cell engineered comprising an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell.

2. The cell of claim 1, wherein IRF1 expression is under control of a constitutive promoter, such as any eukaryotic version of the Cytomegalovirus Promoter, Elongation Factor- 1 Alpha Promoter, Simian Virus 40 Promoter, Phosphoglycerate Kinase Promoter, EF-^Į^ Promoter, CMV-HTLV Hybrid Promoter, CMV Early Enhancer / Chicken Beta-Actin Promoter, Glyceraldehyde 3-Phosphate Dehydrogenase Promoter, U6 promoter, or Heat Shock Protein Promoter.

3. The cell of claim 1, wherein IRF1 expression is under the control of an conditional / inducible promoter, such as a Tet-inducible promoter, lac-inducible promoter, T7 inducible promoter, IPTG-inducible promoter, or Gal-inducible promoter.

4. The cell of any one of claims 1-3, wherein said exogenous transcription cassette is a non-viral vector.

5. The cell of any one of claims 1-3, wherein said exogenous transcription cassette is a viral vector.

6. The cell of claim 5, wherein said viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral (HSV) vector, a baculoviral vector, a vesicular stomatitis virus (VSV) vector, or others.

7. The cell of any one of claims 1-6, wherein the IRF1 coding region is codon optimized.

8. The cell of any one of claims 1-7, wherein said IRF1 is truncated IRF1 but retains the ability to activate expression of IDO1, COX2, IL4I1, PD- / ^^^F3 / $^Į^^) / *^^^DQG^RU^*DOHFWLQ^ 9.

9. The cell of any one of claims 1-8, wherein the cell is a human cell. 4882-8762-3149, v.

110. The cell of any one of claims 1-8, wherein the cell is a non-human mammalian cell.

11. The cell of any one of claims 1-10, wherein the cell is further engineered to express a selectable marker, such as an antibiotic resistance gene product or a fluorescent / chemiluminescent gene product.

12. The cell of any one of claims 1-11, wherein the cell is further engineered to express a suicide gene under control of a conditional / inducible promoter.

13. The cell of any one of claims 1-12, wherein the cell is an hTERT-modified cell or a primary MSC derived from adipose, bone marrow, umbilical chord, or from an induced pluripotent cell (iPSC).

14. The cell of anyone of claims 1-13, wherein the cell expresses indoleamine 2,3- dioxygenase 1 (IDO1) at increased levels as compared to a comparable cell not engineered to expression IRF1, such as with 30% or more IDO1-highly expressing cells at 7 days or more following engineering.

15. The cell of claim 14, wherein the cell expresses IDO1 at increased levels for at least 1 week, at least two 2 weeks, at least three weeks, at least a month, or at least three months.

16. A mesenchymal stem cell / mesenchymal stromal cell treated or engineered to express interferon regulatory factor 1 (IRF1) protein or an mRNA coding for IRF1.

17. The cell of claim 16, wherein said mRNA coding for IRF1 is codon optimized.

18. The cell of claim 16 or 17, wherein said IRF1 is truncated IRF1 but retains the ability to activate expression of IDO1 and / or COX-2.

19. The cell of any one of claims 16-18, wherein the cell is a human cell.

20. The cell of any one of claims 16-18, wherein the cell is a non-human mammalian cell. 4882-8762-3149, v.

121. The cell of any one of claims 16-20, wherein the cell is engineered to express a selectable marker, such as an antibiotic resistance gene product or a fluorescent / chemiluminescent gene product.

22. The cell of any one of claims 16-21, wherein the cell is engineered to express a suicide gene under control of a conditional / inducible promoter.

23. The cell of any one of claims 16-22, wherein the cell is an hTERT-modified cell or a primary adipose-derived cell.

24. The cell of anyone of claims 16-23, wherein the cell expresses indoleamine 2,3- dioxygenase 1 (IDO1) at increased levels as compared to a comparable cell not engineered to expression IRF1, such as with 30% or more IDO1-highly expressing cells at 7 days or more following engineering.

25. The cell of claim 24, wherein the cell expresses IDO1 at increased levels for at least 1 week, at least two 2 weeks, at least three weeks, at least a month, or at least three months.

26. A method of increasing the in vivo half-life of a mesenchymal stem cell / mesenchymal stromal cell comprising (a) engineering the cell to contain an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell or (b) contacting the cell with interferon regulatory factor 1 (IRF1) or an mRNA encoding IRF1.

27. A method of increasing the number of passages a mesenchymal stem cell / mesenchymal stromal cell will sustain comprising (a) engineering the cell to contain an exogeneous transcription cassette that produces interferon regulatory factor 1 (IRF1) in said cell or (b) contacting the cell with interferon regulatory factor 1 (IRF1) or an mRNA encoding IRF1.

28. A method of producing a conditioned medium comprising culturing the cell of any one of claims 1-25 in a basal medium.

29. The method of claim 28, wherein culturing occurs for about 24-72 hours.

30. A conditioned medium produced according to the method of claim 28 or 29. 4882-8762-3149, v.

131. A method of treating an autoimmune disease in a subject comprising administering the cell of any one of claims 1-25 to said subject.

32. The method of claim 31, wherein the autoimmune disease is atherosclerosis, graft versus host disease, multiple sclerosis, Crohn’s disease, Rheumatoid Arthritis, Type 1 Diabetes, Psoriasis, or Acute Respiratory Distress Syndrome.

33. The method of claim 31 or 32, further comprising administering to said subject a second autoimmune disease therapy, such as an anti-inflammatory agent / immunomodulatory agent.

34. The method of any one of claims 31-33, wherein the subject is a human or non-human mammal.

35. The method of any one of claims 31-34, wherein the subject is administered the cell more than one, such as on a chronic basis, such as weekly, monthly, quarterly, semi-annually, or annually.

36. The cell of claim 4, wherein said non-viral vector is a transposon vector such as a Sleeping Beauty, a PiggyBac vector, or others.

37. The cell of any one of claims 1-7, wherein said IRF1 is truncated, and only includes DNA base pairs 0 through 867 of SEQ ID NO: 1 plus a stop codon.

38. The cell of any one of claims 1-7, wherein said IRF1 is truncated, and only includes DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon.

39. The cell of any one of claims 1-7, wherein said IRF1 is truncated, and only includes 5%, 20%, or 50% of the DNA base pairs 0 to 867 and 100% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon.

40. The cell of any one of claims 1-7, wherein said IRF1 is truncated, and only includes 100% of the DNA base pairs 0 to 867 and 5%, 20%, or 50% of the DNA base pairs 867 through 978 of SEQ ID NO: 1 plus a stop codon.

41. The cell of claim 16 or 17, wherein said IRF1 is truncated as in claims 37-40. 4882-8762-3149, v.

142. The method of claims 1-25 wherein the cell(s) are encapsulated in a biomaterial such as alginate.

43. The method of claim 43 where the biomaterial is modified with anti-inflammatory or anti-fibrotic small molecules.

44. The method of claims 1-25 wherein cells are treated before or after engineering with a biochemical stimulus such as budesonide, dexamethasone, 71)Į^^,)1Ȗ^^ / LSRSRO\VDFFKDULGH^ (LPS), IL-^ȕ^^RU^RWKHUV^ 4882-8762-3149, v.1

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