NEW REGULATORY MACROPHAGES AND USES THEREOF

Abstract

The present invention relates to novel immunoregulatory macrophage cells which are useful in the treatment of different immunological and non-immunological diseases and conditions. The cells are characterized by a specific marker and activity pattern which distinguishes them from other cells. The novel immunoregulatory macrophage cells have a high phagocytosing capacity and are capable to suppress the proliferation of T cells. The invention also provides a novel process for preparing immunoregulatory macrophage cells in suspension culture from blood monocytes. The process is amenable to a high degree of automation. In a still further aspect, the invention relates to a pharmaceutical composition comprising the immunoregulatory macrophage cells of the invention.

Claims

1. A process for preparing an immunoregulatory macrophage cell, said process comprising: (a) isolating CD14 positive monocytes from a blood sample of a subject; (b) culturing the monocytes in a culture medium containing (i) M-CSF and/or GM-CSF, and (ii) a CD16 ligand; (c) contacting the monocytes or monocytes-derived cells with IFN-; and (d) obtaining the immunoregulatory macrophage cell from the culture medium, wherein steps (b) and (c) are performed in a container that is agitated to avoid adherence of the cells to the surface of the container.

2. Process of claim 1, wherein step (d) does not include the mechanical detachment of cells from the surface of the container.

3. Process of claim 1, wherein steps (b) and (c) are performed such that not more than 10%, and preferably not more than 5%, of the cells adhere to the surface of the container.

4. Process of claim 1, wherein the culture medium in step (b) comprises human blood serum, such as human AB serum.

5. Process of claim 1, wherein the concentration of M-CSF and/or GM-CSF in step (b) is in the range of 5-100 ng/ml, preferably 20-25 ng/ml.

6. Process of claim 1, wherein the monocytes in step (b) are cultured for at least 3 days, for at least 4 days, for at least 5 days, for at least 6, or for at least 7 days prior to contacting with IFN-.

7. Process of claim 1, wherein the container is made of plastic, preferably ethylene vinyl alcohol (EVOH), ethylene vinyl acetate copolymer (EVA), or polyolefine.

8. Process of claim 1, wherein the concentration of IFN- in step (c) is in the range of 5-100 ng/ml, preferably 20-25 ng/ml.

9. Immunoregulatory macrophage cell obtainable by a process according to claim 1.

10. Immunoregulatory macrophage cell, wherein said cell expresses the following markers: CD16, CD163, and Syndecan-3.

11. Immunoregulatory macrophage cell of claim 10, wherein said cell further expresses at least one of the following markers: CD51, CD11c, CD72, IDO1.

12. Pharmaceutical composition comprising the immunoregulatory macrophage cell of claim 10 or a sub-cellular fraction thereof.

13. Pharmaceutical composition of claim 12, wherein at least 70%, preferably at least 80%, and more preferably at least 90% of the cells in the composition are immunoregulatory macrophage cells of claim 10.

14. A method of suppressing transplant rejection and/or prolonging transplant survival in a subject receiving a transplant, the method comprising administering an immunoregulatory macrophage cell according to claim 10 or sub-cellular fraction thereof to the subject.

15. The method of claim 14, wherein said transplant is an allogeneic transplant.

16. A method of promoting or sustaining the engraftment or effect of regulatory T cell cell-based medicinal products, the method comprising administering an immunoregulatory macrophage cell according to claim 10 or sub-cellular fraction thereof to a subject in need thereof.

17. A method of treating or preventing an autoimmune disease, an inflammatory disease, or a hypersensitivity reaction, the method comprising administering an immunoregulatory macrophage cell according to claim 10 or sub-cellular fraction thereof to a subject in need thereof.

18. The method of claim 17, wherein said autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), scleroderma, Sjgren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritides; ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous small-vessel vasculitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behget's disease, thromboangiitis obliterans, Kawasaki disease, and other large-, medium- or small-vessel vasculitides of autoimmune aetiology; Multiple sclerosis (MS) and neuroimmunological disorders; Type I diabetes, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction, and other autoimmune endocrinological disorders; haemolytic anaemia, thrombocytopaenic purpura and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, pemphigoid and other autoimmune dermatological conditions.

19. The method of claim 17, wherein said inflammatory disease is selected from the group consisting of arterial occlusive diseases, such as peripheral artery occlusive disease (pAOD), critical limb ischaemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or constriction; microvascular angina, also known as cardiac syndrome X; inflammation associated systemic with metabolic disorders, including Type II diabetes and obesity-related metabolic syndrome; dermatological diseases, including eczema.

20. The method of claim 17, wherein said hypersensitivity reaction is selected from the group of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity and mastocytosis.

21. A method of promoting tissue-repair processes by participating in tissue remodelling, tissue regeneration, angiogenesis, vasculogenesis, or prevention/limitation of fibrosis, the method comprising administering an immunoregulatory macrophage cell according to claim 10 or sub-cellular fraction thereof to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0181] FIG. 1 shows a comparison of 23 extracellular marker expressions on Mreg-sc and Mreg-bc cells. (A) Background normalized median fluorescence intensities of each marker are shown in dot blots where each dot represents one batch, i.e. donor. (B) In waterfall plot, with bars repre-senting log-scale fold changes in the channel-normalized Median Fluorescent Intensity (nMFI). nMFI was calculated in R v3.5 by grouping assays and channels, then subtracting the FMO MFI measurement that occurred within that group. (C) 10 EC markers were differently expressed in the two different Mreg products to a statistically significant degree. (D) A representative figure of the gating strategy for macrophage phenotyping. For intracellular analyses, CD45 was replaced with CD33 and a fixable viability dye for the APC channel was used. Where 7-AAD was used, the gating logic was again similar. To assess the degree of non-target cell contamination, the gates were extended to cover small-sized cells, such as lymphocytes.

[0182] FIG. 2 shows the results of the gene expression analysis in different macrophages: (A) IDO1 mRNA expression by RT-qPCR; (B) IDO1 protein expression by flow cytometry; (C) Correlation of IDO1 protein expression with detected mRNA levels; (D) DHRS9 mRNA expression by RT-qPCR. Statistical significance of difference in expression between the two products with p<0.0001 is indicated by ***; p<0.001 ** and p<0.01 *. In panels (A) and (D) the dashed line at Log2[Rq]=0 indicates no change in mRNA expression; values above the line indicate mRNA upregulation and values below the line indicate mRNA down-regulation. In panels (B) and (C) the dashed line at MFI=2 indicates a preliminary detection threshold where IDO1 protein is expressed by macrophages at least two-fold higher than the background fluorescence.

[0183] FIG. 3 shows the separation of regulatory macrophages from M1 and M2a macrophages based on expression of DHRS9 and IDO1 mRNA.

[0184] FIG. 4 is a depiction of the preliminary stability data (n=2) demonstrating that Rotea formulated Mreg cells retain high viability at least up to 48 h when stored at +4 C. (A) Mreg-sc (B) Mreg-bc and (C) heat-treated controls at 0 h, 24 h, and 48 h in 5% HSA plasmalyte. For determination of cell viability and quality, Annexin V Apoptosis kit was used and analyzed by MUSE Cell Ana-lyser.

[0185] FIG. 5 shows that the basal secretome profiles of Mreg-bc and Mreg-sc are very similar. Only IP-10 from the 27 factors measured were differentially secreted to the medium. Estimated concentrations in pg/mL are shown in y-axis.

[0186] FIG. 6 shows the results from the phagocytosis assays using pHrodo Green E. coli. Mreg cells were incubated with E. coli particles for 1 hour and analyzed with flow cytometry. (A) Representative histograms of engulfed particles, measured as pHrodo Green fluorescence intensity, in Mreg-bc. Dashed light-gray plot indicates the control without particles incubated at +37 C., light-gray plot shows the control with particles incubated on ice, and dark-gray plot represents the sample with particles incubated at +37 C. (B) Dot plots of the percentages of the cells that had internalized labelled E. coli particles. Results are depicted as meanSD percentages of pHrodo Green-positive cells within the live CD45-positive population (live CD45+CD3+ for T cells).

[0187] FIG. 7 demonstrates that Mreg-sc cells are able to inhibit T cell (CD3+) proliferation in co-cultures (72 h). Results are shown as meanSD from 3-5 Mreg batches (i.e. 3-5 donors). Statistical significance is shown as * p<0.05, ***p<0.001. The addition of an IDO1 inhibitor (1 mM 1-methyl DL-tryptophan) to the co-culture restored proliferation of the T-cells to the level observed with non-suppressing monocytes. Key: first bar=monocytes; second bar=Mreg-bc; third bar=Mreg-sc.

[0188] FIG. 8 shows the results of CD72 expression in Mreg-bc and Mreg-sc and M0, M1 and M2a macrophages.

[0189] FIG. 9 shows the results from transcriptomic analysis. Mreg-sc, Mreg-bc, M1 and M2a were compared to M0 macrophages. The log 2-fold changes of gene transcripts are presented for the genes ARMH1, CA11, SMARCD3 and HLA-DOA. ARMH1, CA11, SMARCD3 are expressed at the same level in M0, M1 and M2a cells. These genes are >4 fold higher (about >log 2 fold change) expressed in Mregs compared to M0, M1 and M2a. HLA-DOA has a 3-fold higher expression Mregs compared to other macrophages.

[0190] FIG. 10 shows that gene transcripts of SELENOP, RNASE1, C1QC and NRA4A3 can be used as specific markers for Mreg-sc. In Mreg-sc NRA4A3 is downregulated, while SELENOP, RNASE1 are C1QC are upregulated.

EXAMPLES

[0191] The Mregs were manufactured in accordance with current GMP principles for the production of sterile medicinal products. Attention is paid at every processing step that products, materials and equipment are protected against contamination and impurities.

[0192] Peripheral blood monocytes were isolated from leukapheresis products of healthy volunteers collected by accredited Blood collection. Informed consent for apheresis donation was obtained in accordance with the Declaration of Helsinki following to the legal regulations transposing the Directives 2002/98/EC and 2004/23/EC.

Example 1: Enrichment of Monocytes

[0193] For enrichment of monocytes, CD14+ monocytes were isolated with the GMP-compliant, fully-closed LP14 Process in CliniMACS Prodigy (Miltenyi Biotec GmbH) according to manufacturer's instructions (LP-14 System User Manual, issued 2015-03). Prior to the process a small proportion of leukapheresis product was sampled and the total cell count and viability were determined by NucleoCounter NC-200 automated cell counter (ChemoMetec), and percentage of CD14+ cells and CD3+ lymphocytes from all CD45+ white blood cells were determined by flow cytometry. Similarly, after the separation process, the target cell population was checked for its purity, cell recovery and viability.

Example 2: Mreg Differentiation

[0194] For differentiation of the monocytes obtained in Example 1 into Mregs, the monocytes were divided into two different fractions. The first fraction was differentiated into Mregs in suspension culture in a Xuri Cell Expansion System according to the method of the invention, thereby providing Mreg-sc cells. The second fraction was differentiated into Mregs in gas-permeable bags to provide Mreg-bc cells. The key parameters of the two processes are depicted in Table 1 below.

TABLE-US-00001 TABLE 1 Key parameters of manufacturing processes of Mreg cells. Culture Cell vessel & cell Culture shaking Type Bioreactor contact material property Medium Mreg-bc N/A bag, flipping of bag RPMI 1640 polyolefin twice 2 mM Mreg-sc Xuri bag, polyethylene Continuous GlutaMAX vinyl acetate/ wave-type: 1 PenStrep low density D0-D1: 2, 10% Human polyethylene; or 2 rpm AB serum ethylene vinyl D1-D7: 4, 25 ng/ml M-CSF alcohol/linear 4 rpm +bolus of 25 low-density ng/ml IFN- polyethylene on day 6

[0195] Differentiation into Mreg-Bc

[0196] Monocytes were differentiated in gas-permeable MACS GMP differentiation bags (Miltenyi Biotec) as described by [6]. When differentiating monocytes in these bags, Mregs become a semi-adherent and more homogenous cell population than their flask-cultured counterparts. Briefly, 1.810.sup.8 monocytes were seeded in a 3 L differentiation bag in 180 ml (i.e. 110.sup.6 cells/ml) of culture medium. The same medium was used for both processes (Table 1): RPMI 1640 supplemented with 10% Human AB serum (HABS), 2 mM GlutaMAX (Invitrogen, Germany), 100 U/ml Penicillin, 100 g/ml Streptomycin (Invitrogen), and 25 ng/ml recombinant human M-CSF (R&D Systems, Germany). The bag was placed in an incubator with a humidified atmosphere at 37 C., with 5% CO.sub.2. At the next day, the bag was flipped in order to provide more surface for monocytes to attach. The cell density on the bag surface was 0.2710.sup.6 cells/cm.sup.2 (if only one surface, 672 cm 2, was counted) or 0.1310.sup.6 cells/cm.sup.2 (if both surfaces, 1344 cm.sup.2, were counted). On day 6 (18-24 h prior to harvest), human recombinant interferon-gamma (IFN-; Merck, Germany) was added and the bag was flipped once again. During the 7-day culture period, no medium changes were performed.

[0197] At the end of the 7-day culture period, the cells in the bag were vigorously shaken to detach them from the inner surface of the bag. The cells were then transferred by syringe to the centrifugation tubes (harvest 1) after which the bags were flushed with DPBS in order to collect the remaining cells from the bag (harvest 2). The two harvest fractions were kept separate, and the tubes were centrifuged at 300g, at RT, for 10 min. Next, supernatants of the first harvest were collected and stored at 80 C. until Multiplex ELISA analysis. The cell pellets were resuspended to DPBS/medium/final formulation buffer (depending on the subsequent analysis). After determination of the total cell number and viability of both fractions by NC-200, they were pooled or only the harvest 1 fraction was used for further studies. These conventionally formulated Mreg-bc cells were allocated for phenotype characterization by flow cytometry, qPCR, and for further functional assays (see below). The harvested and conventionally formulated Mreg-bc were >85% viable, with recoveries of up to 70%.

[0198] Differentiation into Mreg-Sc

[0199] Monocytes were differentiated in a Xuri Cell Expansion System (GE Healthcare). This is a wave-type of bioreactor, originally developed and validated for expansion of T cell based cellular immunotherapy, and its mechanistic principle is that the cell culture is always in motion. Mreg-sc cells were differentiated in 2 L Xuri Cellbags (cat. no. 29-1054-92; GE Healthcare). For this size of the bag, the final volume range of the culture medium that can be used is 300-1000 mL. Seeding density was 1.010.sup.6 cells/ml (one test run with double the amount of cells was performed) in the medium set out above in Table 1. The final process parameters that were finally established are shown in Table 2 below. More than 10 batches of Mreg-sc cells have been manufactured in accordance with these parameters.

TABLE-US-00002 TABLE 2 Parameters of the Xuri differentiation process Process Step Day Process Parameter/Specification Monocyte seeding 0 1 10.sup.6 CD14+ cells/mL medium (see Table I) density Seeding volume 0 300-1000 mL General culture 0-7 5% CO.sub.2, compressed air, +37 C., conditions gas flow 0.1 L/min Waving 0 angle 2, speed 2 rpm parameters 1-7 angle 4, speed 4 rpm Sampling 6 sampling and IFN- addition (in and IFN- medium, 5% volume of the addition batch volume) via sampling port Mreg-sc harvest 7 cold block under the bag for 5-10 min (horizontal position, no rocking) via harvest line, pump speed 100 rpm until cell suspension reaches the harvest bag; then by gravity (harvest 1) via harvest line add cold 500 ml DPBS-2 % HSA to bag; shake with Xuri at 10 rpm, 6 for 10 min via harvest line, pump speed 100 rpm until suspension reaches the harvest bag; then by gravity (harvest 2)

[0200] During the process development phase and after establishment of the final parameters, harvest 1 and harvest 2 were kept separately for determination of cell viability and recovery to better under-stand the process and to ensure that low quality cells are not taken to further analyses. Additionally, at the beginning, immune-phenotyping was performed separately for these two since it was not known whether Mregs readily in suspension (harvest 1) would exhibit differential pattern of extracellular marker expression from those adhering on bag surface and requiring incubation with cold buffer with increased rocking parameters (harvest 2). For secretome analysis, medium samples were collected from harvest 1. Final analyses (see Mreg-sc and below) were performed from centrifuged, resuspended and pooled harvests.

[0201] From a manufacturing point-of-view the Xuri bioreactor solved many bottlenecks that were associated to manufacturing in several, separate, cell differentiation bags. Table 3 shows the results from the bag-based and Xuri-based differentiation processes. The calculations are based on the assumption that in theory from one leukapheresis product of 0.541-3.5510.sup.9 monocytes can be obtained for further Mreg differentiation (data from >30 LPs processed by Prodigy in our lab); an average monocyte yield has been 0.85010.sup.9. Process recoveries and viabilities are calculated from batches (n=6; today in 2020, >10 batches) manufactured in 2019 after establishing the final process parameters for Xuri. It is worth noticing that in this comparison, the numbers for Mreg-bc were obtained from only one 3 L differentiation bag harvested by an experienced operator. Thus, the data obtained from these small-scale and sub-batch derived Mregs do not fully represent a real full-sized, clinical Mreg-bc batch. For both Mreg types, recovery and viability have been calculated from harvested, centrifuged, and buffer-formulated samples.

TABLE-US-00003 TABLE 3 Comparison between Mreg-sc and Mreg-bc processes Xuri Bag based bioreactor based Parameter (Mreg-bc) (Mreg-sc) Number of monocytes/bag.sup.1 1.8 10.sup.8 1 10.sup.9 Number of bags needed for x monocytes: 5.4 10.sup.8 monocytes 3 1 9.0 10.sup.8 monocytes 5 1 3.4 10.sup.9 monocytes 19 4 (2 L); 1 (10 L).sup.1 Recovery % 77 19 35 8 (mean; SD) n = 6 Viability % 96 3 97 1 (mean; SD) n = 6 Number of Mregs, calculated from 6.55 10.sup.8 2.98 10.sup.8 8.50 10.sup.8 monocytes and recoveries Estimated time (in minutes) taken for actions for a batch size of 8.50 10.sup.8 monocytes Day = 0 seeding 25 10 Day 1-bag flipping/reactor speed 10 1 adjustment Day 6 = Addition of interferon- 25 5 Day 7-Harvesting 50 35 Notes: .sup.1Bag size for Mreg-bc is 3L and for Mreg-sc (for Xuri, also bigger bags available);

[0202] It can be seen that the Xuri bioreactor-based method is associated with a lower recovery of Mreg cells compared to the bag-based approach. The main advantage of the bag-based approach is the recovery amount of the recovered Mregs. On the other hand, the Xuri bioreactor-based method results in a comparatively high quality of Mregs. Further, the process has a higher degree of automatization and does hence not dependent on an idiosyncratic individual operator's skills.

[0203] Being suspension-based, rather than adherent-based, it enables the production of the whole batch in one compartment, i.e., in one bag.

Example 3: Preparation of Comparator Macrophages

[0204] M1 and M2a macrophages were produced with slight modifications to the protocols described in [11] in the same gas-permeable MACS GMP differentiation bags as described above in the context with Mreg-bc. Cells were differentiated for 6 days in the same medium as Mreg-bc, but with 5 ng/ml M-CSF and 20% fetal bovine serum (FBS; Gibco) instead of HABS. On day 6, complete medium exchange was performed and serum concentration reduced to 5% FBS. At the same time, M1 cells were polarized with 100 ng/ml of lipopolysaccharides (LPS) from Escherichia coli (Sigma-Aldrich) and 25 ng/ml IFN-, while M2a cells were polarized with 20 ng/ml of recombinant human IL-4 (R&D Systems). Cells were harvested on day 7 similarly to Mreg-bc.

Example 4: Analysis of Extracellular and Intracellular Markers

[0205] The intracellular staining was performed for indoleamine 2,3-dioxygenase (IDO1). In brief, the cells were stained with Fixable Live-Dead dye and blocked with FcR Blocking Reagent. Then, the cells were stained with CD33-PE. After incubation, the cells were fixed and permeabilized and then blocked again with 10% of FcR Blocking Reagent. Finally, the cells were divided into three reaction mixes and intracellular antibodies were added as follows: Tube 1: IDO1-PerCP/eFluor710, Tube 2: IgGl-PerCP/eFluor710 isotype control or Tube 3: no antibody and analyzed after incubation and washing.

[0206] For extracellular markers, a minimum of 210.sup.4 live cells (defined as CD45-positive, SYTOX Green-negative events) were analyzed with the CytoFLEX S. The initial SSC/FSC gate was set up to exclude most of the debris and dying cells. Live cells were then plotted on histograms each depicting one phenotypic marker. Background levels were set based on CD45+/SYTOX Green+ dual-stained cells in order to account for the background autofluorescence. Initially, the level of non-specific mAb binding was estimated using isotype and fluorochrome-matched, non-specific antibodies (isotype controls). A passed quality control (QC) run preceded each analysis run. Also, target MFI values were set up on an external analysis layout using identical assay parameters and Beckman-Coulter Daily QC Beads to verify the instrument performance.

[0207] The IDO1 signal was then analyzed by flow cytometry from a minimum of 110.sup.4 CD33-positive, Fixable Live-Dead stain-negative events after excluding debris, essentially by the same gating strategy as depicted in FIG. 1D. A CD33 and live/dead-stained isotype control was used to determine the level of background and non-specific fluorescence. A passed QC run on the CytoFLEX S flow cytometer preceded each analysis run.

[0208] Flow cytometric data were analyzed with FCS Express 6 Flow Research Edition (DeNovo Software, Glendale, CA, US). Median fluorescence intensity (MFI) was used as the main parameter to describe the intensity of the phenotypic marker expression. For the extracellular markers, the MFI of the control sample was subtracted from the stained sample in order to obtain a background-adjusted MFI values for each sample. For IDO1, an index of specific MFI/isotype MFI was used.

[0209] The percentage of CD14+ monocytes and CD3+ lymphocytes from all CD45+ white blood cells were determined by flow cytometry from the initial leukapheresis sample, and then again after monocyte enrichment in the Prodigy for purity check. For the phenotypic analysis the cells were stained with SYTOX Green dead cell dye as per manufacturer's instructions. The cells were then split into seven reaction mixes and stained with the markers described in Table 4 below. The cells were blocked with FcR Blocking Reagent (Miltenyi Biotec) in all flow cytometry experiments as per kit instructions.

TABLE-US-00004 TABLE 4 Staining scheme of extracellular phenotype markers of regulatory macrophages Re- action tube BV421 BV510 FITC PE PE/Cy7 APC 1 CD284 CD86 Sytox Gr. CD209 CD45 CD370 TLR4 B7-2 DC-SIGN PTPRC Clec9a 2 CD83 CD38 Sytox Gr. CD85h CD45 CD71 HB15 cADPrh ILT1, PTPRC TfR1, LILRA2 TFRC 3 CD80 CD10 Sytox Gr. CD103 CD45 Syndecan3 B7-1 CALLA ITGAE PTPRC SDC3 MME 4 CD163 CD14 Sytox Gr. CD282 CD45 CD206 HbSR TLR2 PTPRC MRC1 5 CD40 CD16 Sytox Gr. CD51 CD45 VEGFR1 FCGR3A ITGAV PTPRC FLT1 6 (CD3)* CD11c Sytox Gr. CD258 CD45 CD49c CD3E ITGAX LIGHT PTPRC ITGA3 TNFSF14 7 N/A N/A Sytox Gr. N/A CD45 N/A PTPRC BV421: Brilliant Violet 421, BV510: Brilliant Violet 510, FITC: fluorescein isothiocyanate, PE: R-phycoerythrin, APC: allophycocyanin, Sytox Gr .: SYTOX Green dead cell dye (Thermo Fisher Scien-tific). *used to assess T cell contamination in the product. HGNC names are underlined.

[0210] The antibodies that were used for flow cytometry and intracellular staining are depicted in Table below. All antibodies depicted in the above Table were used for extracellular staining of CD14+ monocyte purity or phenotypic characterization of regulatory macrophages, except for the final three antibodies which were used for intracellular staining.

TABLE-US-00005 TABLE 5 Antibodies used for flow cytometry and intracellular staining Antibodies used Clone Cat.no. S APC anti-human CD14 MP-9 345787 BD PE anti-human CD45 J33 A07783 BC Brilliant Violet 421 anti-human HTA125 312811 Bl CD284 (TLR4) Brilliant Violet 421 anti-human CD83 HB15e 305323 Bl Brilliant Violet 421 anti-human CD80 2D10 305221 Bl Brilliant Violet 421 anti-human GHI/61 333611 Bl CD163 Brilliant Violet 421 anti-human CD40 5C3 334331 Bl Brilliant Violet 421 anti-human CD3 OKT3 317343 Bl Brilliant Violet 510 anti-human CD86 IT2.2 305431 Bl Brilliant Violet 510 anti-human CD38 HB-7 356611 Bl Brilliant Violet 510 anti-human CD10 HI10a 312219 Bl Brilliant Violet 510 anti-human CD14 M5E2 301841 Bl Brilliant Violet 510 anti-human CD16 3G8 302047 Bl Brilliant Violet 510 anti-human 3.9 301633 Bl CD11c PE anti-human CD209 (DC-SIGN) 9E9A8 330105 Bl PE anti-human CD85h (ILT1) 24 337904 Bl PE anti-human CD103 (Integrin E) Ber_ACT8 350205 Bl PE anti-human CD282 (TLR2) TL2.1 309707 Bl PE anti-human/rat CD51 (Integrin v5) P1F6 920007 Bl PE anti-human CD258 (LIGHT) T5-39 318706 Bl PE/Cy7 anti-human CD45 HI30 304016 Bl APC anti-human CD370 8F9 353805 Bl (CLEC9A/DNGR1) APC anti-human CD71 CY1G4 334107 Bl APC anti-human CD206 (MMR) 15-2 321109 Bl APC anti-human CD49c (integrin 3) ASC-1 343808 Bl Anti-VEGFR-1 (Flt-1)-APC, human REA569 130-108-930 MB Human Syndecan-3 APC-conjugated polyclonal FAB3539A R&D PE anti-human CD33 P67.6 345799 BD PerCP-eFluor 710 Mouse IgG1 P3.6.2.8.1 46-4714-82 eB K Isotype Control PerCP-eFluor 710 IDO1 eyedio 46-9477-42 eB Monoclonal Antibody Key: S = Supplier. BD = Becton Dickinson Biosciences. BC = Beckman Coulter. Bl = Biolegend. eB = eBiosciences. MB = Miltenyi Biotec. R&D = R & D Systems.

[0211] Results: The results obtained from the marker analysis are depicted in FIG. 1. It can be seen that both Mreg processes consistently yielded relatively homogenous populations of macrophages, with certain distinct differences between the two processes. The Mreg-bc cells displayed a prom-inent down-regulation of multiple activation-associated markers, such as CD38, CD40 and CD80, and elevated levels of markers CD86 and CD71 determined by flow cytometry. The notable factors for the Mreg-sc process were high levels of CD11c, CD14, CD16, CD51, CD163 and syndecan-3. Hence, multiple markers allow for a reliable differentiation between the two macrophage products. Furthermore, the two processes were set apart by the differential expression of IDO1, with markedly higher levels seen in Mreg-sc (FIG. 2B). A direct correlation between the degree of IDO1 mRNA upregulation and IDO1 protein expression was observed (FIG. 2C), although some level of mRNA accumulation is required before IDO1 is detected on protein level.

[0212] As a result, not all batches of Mreg-bc showed expression of IDO1 on protein level even though the mRNA was at least 50-fold upregulated compared to CD14+. Thus, IDO1 expression levels can potentially be used to differentiate between different types of macrophages, and even between Mreg-sc and Mreg-bc.

Example 5: Gene Expression Characterization by RT-qPCR

[0213] For RT-qPCR 510.sup.6 cells were resuspended in 500 l RNAprotect Cell reagent (Qiagen) and frozen at 20 C. Total RNA was extracted from cells using RNeasy Protect Cell Mini Kit (#74624, Qiagen) with QIAshredder disposable cell-lysate homogenizers (#79654, Qiagen). RNA amount was quantified by NanoDrop OD260 measurements. Four pg of RNA from each sample was treated with DNAse I (#DNASE-50 PrimerDesign or #18068015 Invitrogen) according to manufacturer's instruction. After the concentration of the samples was adjusted to 50 ng/l and verified by Qubit Fluorometer (Invitrogen) measurements using Qubit RNA HS Assay Kit (#Q32855, Invitrogen). Relative expression of DHRS9 and IDO1 mRNA was measured by RT-qPCR using TaqPath 1-Step Multiplex Master Mix Kit (#A28526 Applied Biosystems) and QuantStudio 5 Real-time PCR system (Applied Biosystems) according to manufacturer's instruction. Taqman assays were purchased from Applied Biosystems: for Human IDO1 assay number Hs00984148_ml (FAM-MGB), for Human DHRS9 Hs00608375 ml (FAM-MGB) and for Human GAPDH Hs03929097_g1 (VIC-MGB). Expression of GAPDH mRNA was used as an en-dogenous control gene for data normalization. Each amplification reaction contained 50 ng of RNA and was performed in triplicates. Obtained amplification data was analyzed with QuantStudio Design and Analysis desktop Software (Applied Biosystems). Changes in expression were calculated relative to expression of corresponding mRNA in the starting material, i.e. CD14+ monocytes.

[0214] Results: DHRS9 expression was considerably increased in Mreg-bc, Mreg-sc and M2a cells (FIG. 2D). For M1 cells, expression of DHRS9 was significantly lower, with some M1 batches showing downregulation of DHRS9 mRNA when compared to the starting CD14+ monocyte population. Thus, the set of two genes, IDO1 and DHRS9, can assist in identification of regulatory macrophages from M1 and M2a phenotypes. Regulatory macrophages exhibit elevated expression of both IDO1 and DHRS9, while M1 pro-inflammatory macrophages have increased expression of IDO1 only and M2a anti-inflammatory macrophages express DHRS9 only (FIG. 3).

Example 6: Cytokine and Growth Factor Secretion Profiles

[0215] For secretome analysis, cell culture medium samples were collected during harvest. All the samples were stored at 80 C. until analysis with Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad Laboratories, Hercules, CA, USA; #M500KCAF0Y). The kit provides for the detection of the anti-inflammatory factors IL-1ra, IL-4, IL-10, IL-13, the pro-inflammatory factors TNF-, IL-1B, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, RANTES, Eotaxin, MIP-1, MIP-1, MCP-1 [MCAF], INF-, IP-10 and the growth factors PDGF bb, VEGF, G-CSF, GM-CSF, and bFGF. Despite of previous categorization, several of these factors are pleiotropic, and thus, their functions are depending on the affected cell types and the microenvironment where they are expressed.

[0216] The experimental procedure was performed according to the manufacturer's instructions. Briefly, magnetic beads coated with capture antibodies were incubated with premixed standards or sample supernatants on a shaker for 30 min. Then, the detection antibodies were added and incubated as above. After washing, streptavidin-PE was added and incubated for 10 min. After washing, the beads were re-suspended in assay buffer, and results were read on the Bio-Plex 200 system. Data were analyzed with Bio-Plex Manager software version 4.1.1. The secretion profiles, i.e. the secretomes, of non-stimulated (basal level) Mreg-sc and Mreg-bc cells from medium samples collected upon harvest were determined.

[0217] Results: Secretome profiles of Mreg-sc (n=3; produced in 2019 with final Xuri process parameters) and Mreg-bc (n=11, samples collected from batches 2018-2019) were analyzed by BioRad multiplex enzyme-linked immunoassay. From 27 factors that were analyzed, 8 were found differently released from Mreg-bc and Mreg-sc when using a Welch two sample method in R (data not shown). However, further checking, and parallel comparison to secretomes of M1 and M2a macrophages, only secretion of IP-10 (FIG. 5), an IFN- induced protein 10 [13], could be found to be significantly secreted from Mreg-sc but not from Mreg-bc. As a conclusion, the basal secretion profiles of these two Mreg products appear to be very similar to each other.

Example 7: Phagocytosis Assay

[0218] A flow cytometry-based phagocytosis assay was performed with Mreg-sc (cultured in suspension) and Mreg-bc (cultured in bags) to evaluate the functional ability of macrophages. The assay was performed with harvested macrophages and cryopreserved and thawed CD14+ monocytes and CD3+ T cells using the pHrodo Green Escherichia coli (E. coli) BioParticles Phagocytosis Kit for Flow Cytometry (Invitrogen by ThermoFisher Scientific, #P35381) according to the manufacturer's protocol with slight modifications. Briefly, one million macrophages/monocytes that were allowed to adhere for at least 1 hour post-harvest and 0.5 million T cells were incubated with pHrodo Green E. coli BioParticles Conjugate (1:20 dilution in culture medium) for 1 hour at +37 C. as well as on ice in order to inhibit particle uptake. After washing, cells were harvested and suspended in 100 l of FACS buffer. Cells were stained with 5 l of 7-AAD viability dye (BD Biosciences, #559925) and 1 l of CD45-PE/Cy7 (Biolegend, #304015), and additionally, 1 l of CD3-BV421 (Biolegend, #317344) for T-cell samples. After incubation for 15 min at RT in the dark, unbound antibodies were removed by a wash with 2.5 ml of FACS buffer prior to acquisi-tion on the CytoFLEX S flow cytometer (Beckman Coulter, US). A minimum of 410.sup.4 live cells (doublet discriminated CD45-positive, 7-AAD-negative) were acquired. Flow cytometric data were analyzed in the FlowJo software (TreeStar, US). Phagocytosis was determined as an increase in pHrodo Green fluorescence relative to the one observed with control sample, incubated on ice, and calculated as the frequency of pHrodo Green-positive cells that had engulfed particles (percentage positive).

[0219] Results: The results are shown in FIG. 6. Flow cytometry analysis showed that both Mreg types (and monocytes) are actively phagocytosing E. coli (FIG. 6A). The detection of phagocytosis events was confirmed by a negative control that contained target particles and was incubated on ice, thus preventing phagocytosis (FIG. 6A). After 1 hour, 85+4% of Mreg-sc and 75+7% Mreg-bc macrophages (834% monocytes) had internalized particles (FIG. 6B).

Example 8: T Cell Suppression Assay

[0220] The expression of IDO1 was identified as the main culprit of the Mreg suppressive effect on T cell proliferation [6], [11], likely due to induced tryptophan deprivation. As shown above, Mreg-sc express higher levels of indoleamine IDO1 than Mreg-bc both on the mRNA and protein level. Therefore, it was investigated whether Mreg-sc exert a stronger Mreg-mediated suppression of T cell proliferation. Suppression tests were performed according to the protocol described in [11] with minor modifications. One day prior to Mreg harvest CD3+ T cells of allogenic donors were fluorescently labeled with CytoTell Green (22253, AAT Bioquest) according to manufacturer's protocol. CytoTell-labeled and unlabeled control T cells were activated using MACSbeads (in 1 bead per 2 cells; 130-091-441, Miltenyi Biotec). The cells were cultured in RPMI growth medium supplemented with 5% HABS, 1% Glutamax and 100 IU/ml Penicillin and 100 g/ml Streptomycin overnight (+37 C., 5% CO.sub.2) along with non-activated control CytoTell-labeled T cells. Next day, freshly harvested macrophages and thawed cryopreserved monocytes were plated into wells of 48-well plate in the culture medium specified above. Cells were allowed to attach for 2 hours (+37 C., 5% CO.sub.2) prior to addition of T cells. The total amount of seeded monocytes/macrophages varied from 6.2510.sup.4 to 3.7510.sup.5 cells per well, so that upon addition of 1.2510.sup.5 T cells to the wells resulting co-cultures were at 3:1, 2:1, 1:1 and 1:2 effector cells to T cell ratios. To investigate the role of IDO1 in Mreg-mediated suppression of T cell proliferation IDO1 inhibitor (1-methyl DL-tryptophan, 860646, Sigma Aldrich) was added to separately designated 3:1 co-cultures. After 72 h in co-culture (+37 C., 5% CO.sub.2) non-adherent cells (mainly T cells) were harvested and labeled with anti-CD3-APC (300312, BioLegend) to identify T cells. For live/dead discrimination 7-AAD (559925, BD Biosciences) exclusion was used. Extent of T cell proliferation was assessed by flow cytometry on CytoFLEX S. Flow cytometric data were exported as FCS 3.0 files and proliferation analysis was carried out in FCS Express 6 Flow Research Edition to calculate proliferation index (PI) for the viable T cell population (CD3 positive cells, 7-AAD negative). Proliferation index in FCS express represents fold expansion during culture (ratio of final cell count to starting cell count) and is calculated according to formula:

[00001]$\mathrm{PI}=\frac{{.Math.}_0^i\mathrm{Ni}}{{.Math.}_0^i\frac{\mathrm{Ni}}{2^i}}$

where i is the cell generation and N is the number of cells observed in that generation.

[0221] Results: No significant difference in T cell proliferation was observed in 1:2 and 1:1 co-cultures with Mreg-sc, Mreg-bc or monocytes. However, at higher cell ratios, 2:1 and 3:1, T cells co-cultured with both Mreg-sc and Mreg-bc proliferated at a lesser extent than T cells cultured alone (data not shown) or in co-cultures with monocytes (FIG. 7). The observed suppression of T cell proliferation was exacerbated with Mreg-sc. Addition of the IDO1 inhibitor (1 mM 1-methyl DL-tryptophan) to the 3:1 co-culture relieved inhibitory effect and restored proliferation of the T-cells to the level observed with non-suppressing monocytes. This confirmed that the inhibitory effect seen in T cell proliferation is linked to the IDO1 expression level by the regulatory macrophages and not to non-specific nutrient limitation in co-cultures.

LITERATURE

[0222] [1] Hutchinson J A, Riquelme P, Sawitzki B, et al. Cutting edge: immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients. J Immunol 2011; 187:2072-8. [0223] [2] Hutchinson J A, Riquelme P, Brem-Exner B G, et al. Tranplant acceptance-inducing cells as an immune-conditioning therapy in renal transplantation. Transpl Int 2008; 21:728-41. [0224] [3] Hutchinson J A, Brem-Exner B G, Riquelme P, et al. A cell-based approach to the minimi-zation of immunosuppression in renal transplantation. Transpl Int 2008; 21: 742-54. [0225] [4] Hutchinson J A, Roelen D, Riquelme P, et al. Preoperative treatment of a pre-sensitized kidney transplant recipient with donor-derived transplant acceptance-inducing cells. Transpl Int 2008; 21: 808-13. [0226] [5] Hutchinson J A, Govert F, Riquelme P, et al. Administration of donor-derived transplant acceptance-inducing cells to the recipients of renal transplants from deceased donors is technically feasible. Clin Transplant 2009; 23: 140-5. [0227] [6] Hutchinson J A, Ahrens N, Geissler E K. MITAP-compliant characterization of human regulatory macrophages. Transplant International 2017; 30(8), 765-775. [0228] [7] Hummitzsch L, Zitta K, Rusch R, et al. Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro. Stem Cells International 2019; Jun. 25. [0229] [8] de Arajo EF, Medeiros D H, Galdino N A et al. Tolerogenic Plasmacytoid Dendritic Cells Control Paracoccidioides brasiliensis Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner. PLoS Pathogens 2016; 12(12), 1-29. [0230] [9 ] Yun T J, Lee, J S, Machmach K, et al. Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells. Cell Metabolism 2016; 23(5), 852-866. [0231] [10] Zhao Y, Wu T, Shao S, et al. Phenotype, development, and biological function of mye-loid-derived suppressor cells. Oncoimmunology 2016; 5(2), e1004983 [0232] [11] Hutchinson J A, Riquelme P, Geissler, EK, Fndrich, F. Human regulatory macrophages. Methods Mol Biol 2011; 677, 181-192. [0233] [12] Munn D H, Shafizadeh E, Attwood J T, et al. Inhibition of T Cell Proliferation by Macrophage Tryptophan Catabolism. The Journal of Experimental Medicine 1999; 189(9), 1363-1372. [0234] [13] Shanmugam N, Reddy M A, Guha M, et al. High glucose-induced expression of prom-flammatory cytokine and chemokine genes in monocytic cells. Diabetes 2003; 52(5), 1256-1264