Methods for improving regulatory t cell production

Abstract

The present disclosure provides, inter alia, substrates for optimizing induced regulatory T cells (iTregs) induction and suppressive activity, and methods for preparing such substrates and using the same to improve iTregs production. Also provided are methods for treating diseases using the iTregs produced by the methods disclosed herein.

Description

METHODS FOR IMPROVING REGULATORY T CELL PRODUCTIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 558,978, filed on February 28, 2024. The entire content of the aforementioned application is hereby incorporated by reference.FIELD OF DISCLOSURE

[0002] The present disclosure provides, inter alia, substrates for optimizing induced regulatory T cells (iTregs) induction and suppressive activity, and methods for preparing such substrates and using the same to improve iTregs production.GOVERNMENT FUNDING

[0003] This invention was made with government support under grant nos. Al 118669 and CA013696, awarded by the National Institutes of Health, and 2036197, awarded by the National Science Foundation. The government has certain rights in the invention.BACKGROUND

[0004] Regulatory T cells (Tregs) are a specialized subset of suppressive cells that promote immunotolerance and avert autoimmune diseases. Conversely, deficiencies in Treg function are linked to a range of diseases; autoimmunity is often associated with a reduced frequency of Tregs (1-11) and compromised suppressive capacity (5, 12-19). The ability to remodulate Tregs through adoptive therapy is thus a compelling strategy in medicine. Early Treg clinical trials mainly focused on graft rejection and graft-versus-host disease (GvHD) (20), where Treg therapies lowered the incidence of acute GvHD (21 , 22), relieved the symptoms of chronic GvHD (23,24), and promoted recovery in patients with severe hyperinflammatory COVID (25). In other disease models like Type 1 Diabetes and Systemic Lupus Erythematosus, adoptive transfer of Tregs transiently stabilizes the disease conditions (26-29). However, many challenges remain in the production of functional and stable Tregs necessary for adoptive therapy.

[0005] Current clinical trials predominately utilize polyclonal thymic Tregs (tTregs), which are generated in vivo from immature thymocytes in response to selfantigens (30). However, reduced functionality of tTregs from patients with autoimmune diseases as well as low frequency of these cells from donors limits the sourcing of these cells for therapy (31). Conventional CD4+T cells, on the other hand, are much more accessible, and can be induced ex vivo into Tregs (iTregs). Cells produced in vitro via the TGF- -dependent pathway were reported to be as suppressive as Tregs induced in vivo (32), and an initial clinical trial has demonstrated the safety of iTregs in adoptive therapy (33). Lastly, tTregs and iTregs shared different TCR repertoires, providing additional freedom in directed response (34). Induced Tregs are thus a critical subset of Tregs with much promise for adoptive transfer immunotherapy.

[0006] Accordingly, there is a need for developing methods for improving regulatory T cell production. This disclosure is directed to meeting these and other needs.SUMMARY

[0007] The present disclosure focuses on using biomaterial design to enhance production of iTregs. In particular, the present disclosure investigates how controlling the mechanical stiffness of materials used to activate T cells can direct subsequent induction long-term for potential clinical application. Previous studies demonstrated that this approach can increase the number of iTregs produced during induction in both humans (polyacrylamide gels) and mice (polydimethylsiloxane, PDMS) (35, 36). Nonetheless, those studies were limited predominantly to short-term (3-day) measures of Treg induction. Studies with other cell systems have shown that mechanosensing can affect epigenetic profiles for various other cell types (37-39)suggesting that a similar effect may occur in iTregs (40, 41). The present dislcosure thus identified how T cell mechanosensing can optimize long-term production of stable and suppressive human iTregs.

[0008] Accordingly, one embodiment of the present disclosure is directed to a substrate for optimizing iTreg induction and suppressive activity having a Young’s Modulus in the range from 15 kPa to 3000 kPa.

[0009] Another embodiment of the present disclosure is a process for preparing a substrate disclosed herein. This process comprises the steps of: (a) mixing an elastomer with a curing agent at a desired ratio; (b) degassing and curing the mixture; and (c) coating the cured mixture with at least one antibody.

[0010] Another embodiment of the present disclosure is a substrate made by a process disclosed herein.

[0011] A further embodiment of the present disclosure is a method for producing iTregs in clinically useful quantities and with clinically relevant suppressive activity. This method comprises the steps of: (a) producing a substrate by a process disclosed herein; (b) culturing regulatory T cells with an induction media on the substrate produced; and (c) collecting the induced cells after a sufficient period of time and repeating step (b).

[0012] Still another embodiment of the present disclosure is a method for treating or ameliorating the effects of a disease in a subject in need thereof. This method comprises administering to the subject an effective amount of iTregs made by a method disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figures 1A-1 D show PDMS substrates for Treg induction. Figure 1A shows the indentation study of PDMS with different ratios of cross-liner and monomer (Data are mean, whiskers are min and max, n=2 individual experiments for 1 :59, n=4 for 1 :10, and n = 5 for 1 :25, and 2 replicates each, One-way ANOVA). Figure 1 B shows fluorescence intensity of PDMS surface coating imaged with an epifluorescence microscope in 40X (Data are mean, whiskers are min and max, n=3 individual experiments and 2 replicates each, One-way ANOVA). Figure 1C showsTreg induction experimental setup. Figure 1 D shows representative gating setup for all samples. Unstained control was used for gating FOXP3+T cells.

[0014] Figures 2A-2D show the impact of substrate stiffness on Treg induction. Figures 2A and 2B show the comparison across 3 different PDMS stiffnesses, based on percent (Figure 2A) and total count of FOXP3+cells (Figure 2B). Figures 2C and 2D show the comparison between softest PDMS (17kPa) surface and stiff controls of plate-bound OKT3 and 9.3 and Dynabeads (2 beads per cell) based on percent (Figure 2C) and total count of FOXP3+cells (Figure 2D). For all panels, Data are mean ± SEM, n=5 individual experiments and 2 replicates each. Two-way ANOVA with Tukey multiple comparisons; * P < 0.05, ** P < 0.01 , *** P < 0.001 , **** P < 0.0001.

[0015] Figures 3A-3B show that mechanosensing in Treg induction relies on cell contractility. Effect of Y-27623 inhibition of actomyosin contractility based on percent (Figure 3A) and total count of FOXP3+cells (Figure 3B). Data are mean ± SEM, n=5 individual experiments and 2 replicates each; Two-way ANOVA with Tukey multiple comparisons; * P < 0.05, *‘ P < 0.01 , *** P < 0.001 , **** P < 0.0001.

[0016] Figures 4A-4B show suppressive capacities of Treg on different PDMS substrates. Figure 4A shows the percent suppression with 1 :1 , 1 :2, and 1 :4 ratios of Treg and Tconvs on 3 PDMS substrates, and gating was created based on unstimulated control. Figure 4B shows the percent suppression with 1 :1 , 1 :2, and 1 :4 ratios of Treg and Tconvs comparing 870kPa to plate-bound control and Dynabeads. One-way ANOVA was conducted with GraphPad Prism, and 870kPa is the control group for comparison with Dynabeads and Plate-bound control for Figure 4B. Data are mean, whiskers are min and max, n=3 individual experiments; * P < 0.05, ** P < 0.01 , “* P < 0.001.

[0017] Figures 5A-5F show that substrate stiffness impacts induced Treg epigenetic modification. Figure 5A shows the PCA analysis of samples epigenetic modification profilers. Figure 5B shows the Volcano plot of differentially methylation analysis comparing tTregs vs Tconvs. Cutoffs are 5% methylation difference, q-value <0.05. Figure 5C shows the Volcano plot of differentially methylation analysis comparing 2600kPa vs 17kPa. Cutoffs are 1 % methylation difference, q-value <0.05.Figures 5D, 5E and 5F respectively show the percent DNA methylation level for all samples on ctla-4, cd254, FoxP3.

[0018] Figure 6 shows contour plots of SSC-A and FSC-A for PDMS substrate to assess T cell activation. Gates were set on the cell population outside of the main population of unstimulated sample.

[0019] Figures 7A-7C show stiffness dependent Treg induction. Figure 7A shows the Median Fluorescence Intensity (MFI) of FOXP3+cells, activated on PDMS of the indicated stiffness, on D3 and D6. Figures 7B and 7C show the comparison between 870 kPa PDMS substrate and rigid controls based on Percent (Figure 7B) and Total count of FOXP3+cells (Figure 7C). Data are mean ± SEM., n =5; * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001 , two-way ANOVA with Tukey multiple comparisons.

[0020] Figures 8A-8B show that substrate stiffness modulates T cell suppressive function. Figure 8A shows representative CFSE data comparing 2600kPa with controls. Figure 8B shows the percent suppression with 1 :1 , 1 :2, and 1 :4 ratios of Treg and Tconvs comparing 2600kPa to plate-bound control and Dynabeads. Data are mean, whiskers are min and max, n=3 individual experiments; One-way ANOVA; * P < 0.05, ** P < 0.01 .

[0021] Figures 9A-9B show the differential methylation analysis on Treg epigenetic profiles. Figure 9A shows Volcano plot of differentially methylation analysis comparing 2,600kPa and 2,600kPa PDMS treated with Y-27632. Figure 9B shows Volcano plot of differentially methylation analysis comparing Tconvs to platebound control.DETAILED DESCRIPTION OF THE DISCLOSURE

[0022] Induced Tregs (iTregs) have great promise in adoptive immunotherapy for treatment of autoimmune diseases. The present disclosure investigates the impacts of substrate stiffness on human Treg induction, providing a powerful yet simple approach to improving production of these cells. Conventional CD4+human T cells were activated on materials of different elastic modulus and cultured under suppressive conditions. Enhanced Treg induction was observed on softer materialsas early as 3 days following activation and persisted for multiple weeks. Substrate stiffness also affected epigenetic modification of Treg specific genes and Treg suppressive capacity. Tregs induced on substrates of an optimal stiffness balance quantity and suppressive quality.

[0023] Accordingly, one embodiment of the present disclosure is directed to a substrate for optimizing iTreg induction and suppressive activity having a Young’s Modulus in the range from 15 kPa to 3000 kPa. In some embodiments, the substrate has a Young’s Modulus of 870 kPa.

[0024] As used herein, “Young’s Modulus” is a mechanical property of solid materials that measures the tensile or compressive stiffness when the force is applied lengthwise. It is the modulus of elasticity for tension or axial compression. As used in the present disclosure and in the art, Young’s Modulus is defined as the ratio of the stress (force per unit area) applied to the object and the resulting axial strain (displacement or deformation) in the linear elastic region of the material. It should also be understood that the substrate disclosed herein may be described by various measures that is known to one skilled in the art. For example, the substrate may be alternatively described by its bulk modulus and / or shear modulus. Such a substrate is encompassed within the scope of the present disclosure, as long as it has a meaningful and measurable Young’s Modulus, which falls within the range disclosed herein.

[0025] Another embodiment of the present disclosure is a process for preparing a substrate disclosed herein. This process comprises the steps of: (a) mixing an elastomer with a curing agent at a desired ratio; (b) degassing and curing the mixture; and (c) coating the cured mixture with at least one antibody.

[0026] In some embodiments, the process comprises attaching the cured mixture to at least one antibody in step (c).

[0027] As used herein, an “elastomer” refers to a polymer with viscoelasticity (both viscosity and elasticity) and with weak intermolecular forces, generally low Young’s Modulus and high failure strain compared with other materials. In some embodiments, the elastomer is selected from the group consisting of aliphatic polyesters, polyacrylamide, polyhydroxyalkanoates, polyurethanes, polyalkylene oxides, siloxanes, polyvinylalcohol, polyvinylpyrrolidone, polylysine, collagen, gelatin,laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides, polysaccharides, and combinations thereof. In some embodiments, the siloxanes can be cyclic or linear.

[0028] In some embodiments, the elastomer is hydrophobic.

[0029] In some embodiments, the elastomer is polydimethylsiloxane (PDMS).

[0030] As used herein, a “curing agent” refers to a substance that is able to participate in the chemical reaction between, e.g., the oligomer, pre-polymer and polymer, to achieve the polymerization process. A curing agent is used to harden a surface or material. It is typically applied to polymer surfaces to facilitate the bonding of the molecular components of the material. The stronger the molecular bonds are, the harder the material surface is. In some embodiments, the curing agent is a crosslinking agent selected from the group consisting of pentaerythritol, (3- Aminopropyl)triethoxysilane (APTES), (3-Aminopropyl)trimethoxysilane, 3- Aminopropyl-methyl-diethoxysilane, 3-(Dimethoxymethylsilyl)propylamine, tetraacetoxysilane, triacetoxy methylsilane; triactetoxy ethylsilane, tetraethyl silicate, and combinations thereof.

[0031] As disclosed herein, the stiffness of the substrate produced can be modulated by controlling the ratio of the curing agent to the elastomer. In some embodiments, the desired ratio is a weight ratio of the curing agent and the elastomer in the range from 1 :10 to 1 :60.

[0032] Another embodiment of the present disclosure is a substrate made by a process disclosed herein.

[0033] A further embodiment of the present disclosure is a method for producing iTregs in clinically useful quantities and with clinically relevant suppressive activity. This method comprises the steps of: (a) producing a substrate by a process disclosed herein; (b) culturing regulatory T cells with an induction media on the substrate produced; and (c) collecting the induced cells after a sufficient period of time and repeating step (b). In this method, iTregs can be collected continuously.

[0034] In some embodiments, the method comprises collecting the induced cells and repeating step (b) every 3 days for up to 15 days.

[0035] In some embodiments, the substrate produced in step (a) has a Young’s Modulus in the range from 15 kPa to 3000 kPa. In some embodiments, the substrate produced in step (a) has a Young’s Modulus of 870 kPa.

[0036] As used herein, an “induction media” refers to any media that is able to induce the regulatory T cells. It includes all conventional and potential media that is currently used or under development in the art. In some embodiments, the induction media is selected from the group consisting of TGF-p, retinoic acid, rapamycin, IL-2, or combinations thereof.

[0037] Still another embodiment of the present disclosure is a method for treating or ameliorating the effects of a disease in a subject in need thereof. This method comprises administering to the subject an effective amount of iTregs made by a method disclosed herein.

[0038] In some embodiments, the disease is selected from the group consisting of an autoimmune disease, an inflammatory complication, and a graft- versus-host disease.

[0039] As used herein, an “autoimmune disease” or “autoimmune disorder” refers to a condition that results from an anomalous response of the adaptive immune system, wherein it mistakenly targets and attacks healthy, functioning parts of the body as if they were foreign organisms. An autoimmune disease or disorder can be organ-specific or non-orgen-specific. Non-limiting examples of an autoimmune disease or disorder include Type I diabetes, Grave’s disease, inflammatory bowel disease, multiple sclerosis, psoriasis, Rheumatioid arthritis, scleroderma, system lupus erythematosus.

[0040] As used herein, an “inflammatory complication” or “inflammatory condition” refers to diseases in which excess inflammation is the key. These diseases can affect different parts or systems in the body, including, e.g., digestive tract and immune system.

[0041] As used herein, a “graft-versus-host disease” or “GvHD” is a systemic disorder that occurs when the graft’s (i.e. , the transplanted) immune cells recognize the host (i.e., the tissues of the recipient) as foreign and attack the recipient’s body cells.

[0042] In some embodiments, the disease is selected from the group consisting of chronic GvHD, hyperinflammatory COVID, Type 1 Diabetes, and Systemic Lupus Erythematosus.

[0043] As defined above, in the context of the present disclosure, a “subject” is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present disclosure include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. In some embodiments of the present disclosure, the phrase “a subject” means a subject having a disease such as chronic GvHD.

[0044] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present disclosure may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because not every treated subject may respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

[0045] As used herein, the terms “ameliorate”, "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

[0046] In the present disclosure, an "effective amount" of a therapeutic is an amount of such therapeutic that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate ofexcretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of the subject, and like factors well known in the arts of, e.g., medicine and veterinary medicine. In general, a suitable dose of a therapeutic according to the disclosure will be that amount of the agent, which is the lowest dose effective to produce the desired effect with no or minimal side effects. The effective dose of a therapeutic according to the present disclosure may be administered as a single dose, or two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the treatment course (e.g., not necessarily on the same day).

[0047] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0048] The following examples are provided to further illustrate the methods of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.EXAMPLESExample 1 Materials and Methods

[0049] PDMS fabrication. PDMS(Polydimethylsiloxane) slabs were fabricated from conventional Sylgard 184 (Dow Corning). Weight ratios of curing agent to elastomer base were varied to generate stiffness of substrate (1 :10, 1 :25, 1 :59). PDMS mixture of monomer and cross-linker was spun at 3000g for 3 min, then poured in 24-well culture plate. These substrates were degassed and cured overnight at 65°C. Substrates (including tissue culture treated plate) were coated with 50 pg / ml of (1 :4) anti-CD3 (clone OKT3, BioXcel) and anti-CD28 (clone 9.3, BioXcel) for 2 hours at room temperature (RT, 23°C).

[0050] PDMS surface coating characterization. PDMS was fabricated as mentioned above but poured onto a glass coverslip with a silicone ring gasket of the same dimensions as a 24-well plate. After curing, PDMS was coated with anti-CD3and anti-CD28 antibodies conjugated with Alexa Fluor 568 (Biolegend). PDMS was imaged with an epifluorescence microscope (Olympus IX83), and fluorescent intensity was quantified using Imaged.

[0051] PDMS stiffness characterization. The Young’s modulus (E) of prepared gels was measured by indentation (48). Slabs of PDMS were deformed with a flat cylindrical head, using a calibrated mass. The material’s Young’s modulus was estimated from the head diameter (D, 8 mm), deflection (h), weight (m), gravitational constant (g), and Poisson ratio (v) of 0.5 assuming Hertzian contact;

[0052] Cell isolation and culture. PBMCs (Peripheral Blood Mononuclear Cells) were isolated from Leukopacks (New York Blood Center) using a Ficoll density gradient centrifugation technique. CD4+CD25_T cells were isolated from PBMCs using a human CD4+CD25+Regulatory T Cell Isolation Kit from Miltenyi in a two-step negative isolation where non-CD4 cells were first depleted and then CD25+cells removed. CD4+CD25_T cells were then frozen in 40% Fetal Bovine Serum (FBS) and 10% DMSO in complete cell culture media, consisting of RPMI 1640 supplemented with 10 mM HEPES, 10 mM L-glutamine, 10% fetal bovine serum, 0.34% p-mercaptoethanol, and 10 mM penicillin-streptomycin. Standard culture conditions were 37°C, humidified 5% Ct / balance air environment.

[0053] Treg induction. 500k cells / ml of cells were seeded on coated PDMS with 10 ng / ml of IL-2 (PetroTech) and TGF-P (PetroTech) and 1 mM of sodium pyruvate and MEM non-essential amino acids for 72 hours (35). Dynabeads (2 beads per cell, Thermofisher) and unstimulated samples served as the positive and the negative control, respectively. After incubation, T cells were removed from the substrates with gentle pipetting. Cell solutions were counted with hemocytometer with Trypan blue dye, fixed and permeabilized with a True-Nuclear™ Transcription Factor Buffer Set following the manufacturer’s protocol for staining (Biolegend). Cells were then reseeded in the concentration of 500k cells / ml supplemented with media containing 10 ng / ml of IL-2. This process was repeated every 3 days for up to 15 days. Cells were restimulated with Dynabeads on Day 9.

[0054] Staining and flow cytometry. FOXP3 expression was assessed with Alexa Fluor 488 anti-human FOXP3 antibodies (Biolegend, 320111) following the manufacturer’s protocol with a staining concentration of 1 :20 at RT for 30 min. Data was collected using a FACSCanto II. Live sorting of (CD4+CD25+CD127_) cells was performed with BB Influx cell sorter. The live Tregs were stained with CD4 antibody in BV421 (1 :20, Biolegend, 317434), CD25 antibody in APC (1 :50, Miltenyi, 130-113- 284), and CD127 antibody in FITC (1 :20, Biolegend, 351312) at 4 °C for 20 min before sorting. All analysis was performed on FCS Express V6 (De Novo). Dead cells were excluded only from analysis by forward / side scatter gating. Doublets were excluded as well. The rest of cells were then used for further analysis.

[0055] Treg inhibitor studies. Y-27632 (Sigma-Aldrich) was dissolved in filtered DI water, and a final concentration of 15 pM was used during seeding.

[0056] Treg Suppressive assay. T cells from day 3 were continued in culture supplemented with IL-2 (no TGF-P). On day 6, live cells were collected to sort for the CD4+CD25+CD127‘ population with BB Influx. After overnight resting in 10 ng / ml of IL-2, Treg suppressive assay was set up according to an established protocol (60). Tresponders (Conventional T cells, Tconvs) were thawed on day 6 and rested overnight. Tregs:Tresponders ratios of 1 :1 , and 1 :2 were utilized to assess Treg functionality. Tregs were counted and suspended at 1 million cells / mL. The initial concentration of 50 k Tregs in 100 pL was seeded on a 96 well-round bottom plate, then serial dilution was performed with 50 pL of media. 25 k cells of Tconvs stained with CSFE in 50 pL were added to each well. 100 pL of Dynabeads in media were then included in the culture, generating a 1 :3 ratio of Tconvs to Dynabeads. Tconvs stimulated with Dynabeads were used as a T cell activation control, and the Tconvs sample alone was used as unstimulated control. Dynabeads were detached after 3 days of induction, then flow cytometry was performed with BD Cantoll to measure CSFE (ThermoFisher) fluorescence intensity. The percent suppression was analyzed with FCS express by creating a marker excluding unstimulated control and calculated based on an established protocol (61 ).

[0057] Targeted NextGen Bisulfite Sequencing (tNGBS). Targeted NextGen Bisulfite Sequencing was carried out by EpigenDx on fixed and permeabilized FOXP3+Tregs sorted with BB Influx after 72 hours of induction. Sample digestion, bisulfite modification, multiplex PCR, library preparation,sequencing, and alignment were carried out by EpigenDx for these assays. Methylation levels were calculated by dividing the number of methylated reads by the total number of reads. The genes of interest and locations of CPG sites can be found in the supplementary materials.

[0058] Computational Analysis. Analysis of methylation data was conducted with the R package methylkit as described in (62). Sample coverages were normalized across samples with the method “mean”. Differential methylation analysis was conducted with either Fisher’s exact test or logistic regression depending on the number of replicates comparing 2 samples. Q values lower than 0.05 were considered significant. Volcano plots were plotted with the package EnhancedVolcano.

[0059] Study approval. Primary human T cells were isolated from blood products from healthy donors (New York Blood Center, NYBC). These products were provided without donor-identifying information. As the samples were deidentified, this study is exempt from DHHS regulations based on §46.104(d)(4).

[0060] Statistical analysis. Data analyses were performed with two-way ANOVA (anovan) in MATLAB with Tukey multiple comparisons. One-way ANOVA were performed with GraphPad Prism 9 (GraphPad Software). P-values less than 0.05 was considered statistically significant (a=0.05).

[0061] Data Availability. Data will be made available on request.Example 2PDMS as a platform to study mechanosensing of Treg induction

[0062] The effect of mechanosensing regulatory T cell induction was examined using planar substrates of polydimethylsiloxane elastomer (Sylgard 184, PDMS) (36, 42). Stiffness was modulated by controlling the ratio of cross-linker to base elastomer (1 :10, 1 :25, 1 :59), producing substrates with Young’s modulus of 17kPa, 870kPa, and 2600kPa (Figure 1A). For Treg induction, these substrates were coated with activating antibodies to CD3 and CD28 (clones OKT3 and 9.3, respectively). The per-area amount of activating proteins on these surfaces wascompared by microscopy of substrates coated with fluorescently labeled OKT3 and9.3 and found to be similar across formulations (Figure 1 B).Example 3 Treg induction on PDMS is mechanosensitive

[0063] Primary human CD4+CD25_T cells (Tconvs) were seeded onto prepared PDMS surfaces in the presence of TGF-p and IL-2 (Figure 1C). Differences in Treg induction, measured by expression of FOXP3 (43) a master regulator of Treg development and function (44), were observed as early as 3 days after activation (Figure 1 D). As shown in Figures 2A and 2B, both the percentage and total number of FOXP3+cells (from a starting population of 1x106Tconvs) increased with decreasing rigidity. T cell activation (as measured by cell size and granularity) was high (~ 100%) and consistent across all PDMS substrates (Figure 6), thus Treg induction is a function of stiffness and not simply differences in activation. The fluorescence intensity (Fl) of FOXP3 induced on PDMS also showed mechanosensing responses, with median Fl within the FOXP3+cells on D3 increasing with decreasing PDMS stiffness (Figure 7A).

[0064] To confirm that cells are responding to the mechanical stiffness of the substrates, 3-day induction was carried out in the presence of Y-27632, the most widely used toward mechanosensing pathway. It inhibits cytoskeletal contractility via ROCK (45). Treatment of cells with Y-27632 reduced the differences in both percentage and total number of FOXP3+cells between substrates (Figures 3A and 3B), indicating that force generation is central to cell response to these materials. Notably, the Y-27632 treatment did not eliminate FOXP3 expression, indicating that inhibiting contractility did not simply shut down cell function.Example 4Treg induction shows persistent mechanosensitive responses over multiple weeks

[0065] For immunotherapy applications, Tregs are normally expanded over the course of multiple weeks, much longer than the initial time point presentedabove. To examine these timescales, T cells were diluted to a density of 5 x 105cells / mL with media containing IL-2 every third day following day 3. Cells were restimulated with Dynabeads on Day 9 to maintain proliferation after the cells had come to rest to isolate the effect of initial stimulation. As shown in Figure 2A, the initial impact of substrate stiffness on Treg induction continued over the entire course of growth. That is, the softest substrate produced the highest percentages and counts of FOXP3+T cells observed on Days 6 and 9 (Figures 2A and 2B). The difference between 870kPa and 17kPa was not observed on Day 12 and Day 15, possibly a result of restimulation of cells from all samples on Day 9 with Dynabeads. With regards to total counts, around 60 million Tregs (60-fold-expansion) were generated at the end of D15 for both 870kPa and 17kPa, but only approximately 44 million Tregs were induced on 2600kPa PDMS substrate. These results are very promising for two reasons: 1) Treg counts were effectively modulated by substrate stiffness, and 2) even after initial activation and induction, this difference in Treg counts was still observed over the course of two weeks.

[0066] Long-term induction and expansion of Tregs using the softest PDMS substrate (17kPa) were also compared to Dynabeads, a widely used platform for cell activation consisting of stiff plastic particles. However, we could not ensure that the antibodies against CD3 and CD28 are identical between the PDMS substrates and Dynabeads (which present a proprietary mix of antibodies). Moreover, the difference in substrate geometry may influence Treg induction. As a more directly comparable substrate, tissue culture plastic coated with OKT3 and 9.3 was also included as a stiff control substrate. The percentage of Tregs induced on the 17kPa substrate was higher than the plate-bound control until Day 15 (Figure 2C), and total Treg counts were always higher on the softest PDMS (Figure 2D). On the other hand, the 17kPa substrate induced a higher rate of Tregs compared to Dynabeads on Day 6, and the difference was detected again after restimulation on D9 (Figure 2C). In terms of total Treg counts, the 17kPa substrate outperformed the Dynabeads until D9, and there was no difference between them on D15 (Figure 2D). Induction on PDMS of intermediate stiffness (870 kPa) showed smaller effects than the softest PDMS, and are included in Figures 7B and 70. These results further support the observation that with the same activating proteins, mechanically softer surfaces yield higher iTreg production. Combining substrate rigidity with further optimization of antibodycomposition may improve production of iTregs potentially outperforming the particlebased Dynabeads.Example 5Stiffer substrates induce Tregs with higher suppressive capacity

[0067] We next evaluated the functionality of iTregs produced using substrates of varying stiffness, comparing their ability to inhibit division of conventional T cells. Target Tconvs were stimulated with Dynabeads in the presence of CD4+CD25+CD127_iTregs collected 6 days after initial activation, and their division over 3 days was measured using a CFSE dye-dilution assay (Figure 8A). In the absence of iTregs, most of Tconvs exhibited at least one division. By contrast, the presence of iTregs produced on PDMS substrates reduced Tconv proliferation (Figure 4A). Notably, iTregs produced on the softest substrates (17 kPa) were less effective in inhibiting target cell proliferation than those produced on stiffer counterparts at iTreg: Tconv ratios of 1 :1 and 1 :2 (Figure 4A); while softer substrates produce more FOXP3+T cells, this is associated with a decrease in functionality. The intermediate stiffness of 870 kPa was thus identified as an optimal condition that balances cell number against functionality. Cells produced on this optimized surface were more functional than those activated using rigid, plate bound OKT3 and 9.3, further demonstrating the mechanosensing response, as well as counterparts from Dynabeads controls (Figure 4B).Example 6 Induced Treg’s epigenetic profiles are stiffness-dependent

[0068] As a complementary view of long-term impacts of substrate stiffness, we compared epigenetic modifications of Treg-associated genes. Targeted Next- Gen Bisulfite Sequencing was carried out on FOXP3+iTregs after 3 days of induction to compare methylation of F0XP3, IL-2RA (CD25), CTLA-4 (CD152), TNFRSF18 (GITR), IKZF2 (Helios), and 1KZ.F4 (Eos). Samples included cells induced on each of the three PDMS formulations, coated plastic, and Dynabeads, as well as Tconv and tTreg cells isolated from the PBMC samples. While PCA analysis of CpG methylation showed the largest difference between tTregs and the rest of the samples alongPrincipal Component (PC) 1 (-60% of variance, Figure 5A), iTregs also separated along a second axis (PC2, -10% of variance), as a function of substrate stiffness and composition. Cells induced using the stiff 2,600 kPa surface were distinct from those produced using the soft 17 kPa substrate and furthest separated from Tconvs along this axis (Figure 5A). In addition, treatment of cells induced using the 2,600 kPa substrate with the cytoskeletal contractility inhibitor Y-27632 was similar to those activated on the softer PDMS materials, indicating substrate resistance to cellgenerated forces alters the methylation state of iTregs.

[0069] Differential methylation analysis supported the insight from PCA and provided additional detail. Large differences in methylation were detected between tTregs and Tconvs in CPG islands from CTLA4, F0XP3, IL2RB, IL2RA, IKZF2, TNFRSF18, and TNFRSF25 (Figure 5B, q-value <0.05). The q-value is a p-value that has been adjusted for the False Discovery Rate (FDR) with the SLIM method for the validation of statistical significance (46). Subtler differences were observed between Tregs produced using 2600kPa and 17kPa PDMS (Figure 5C, q-value <0.05). Moreover, Y-27632 treatment of cells induced using the hard 2600kPa substrate produced similar differential methylation analysis as that between the hard PDMS and the soft PDMS (26 common genes) (Figure 9A); inhibition of actomyosin contractility makes the stiffer substrate respond to stimulus more like the softer substrate, not just in the percent of FOXP3+cells and Treg counts, but also in epigenetic modification.

[0070] We observed a collective of Treg-specific genes that were significantly more hypomethylated on the harder substrate, rendering more tT reg-like epigenetic profiles (41). In particular, our data revealed that exon2 on CTLA4 is hypomethylated for cells induced on stiffer substrates (2,600 kPa) than their softer counterparts (Figure 5D). Exon2 is considered to be a Treg-specific region defining cell commitment and stability upon activation. Induction on the stiffer substrates also resulted in more tT reg-like methylation for PD-L1(CD254) (Figure 5E), IL2RB, PD-L2 (PDCD1LG2), and TNF. Finally, methylation analysis revealed stiffness dependent modulation in 3 CpG islands of the CNS2 region of FoxP3.Example 7Discussion

[0071] Inspired materials engineering was a central feature in making T cellbased therapies clinically deployable. Solid bead supports presenting ligands to CD3 and CD28 for T cell activation, such as the Dynabeads system, provided a manufacturable alternative to antigen presenting cells. Replacing the rigid polystyrene of Dynabeads with mechanically softer PDMS elastomer improves activation and expansion of T cells (42, 47). Subsequent developments include the use of fibers and other alternative formats to provide cytokines and more physiological presentation of ligands, improving T cell expansion. We note that T cells also respond to the mechanical stiffness of other materials used extensively in fundamental studies of cellular mechanobiology. In particular, our group previously demonstrated that T cell activation and Treg induction are sensitive to the stiffness of polyacrylamide hydrogels (35, 48). However, with a focus on T cell production, this report uses PDMS, which has advantages of stability and regulatory history compared to polyacrylamide gel. In addition, our data demonstrates that PDMS provides better short-term (3 day) induction of Tregs (up to 60%) compared to polyacrylamide (30%) as measured by percentage of FOXP3+cells. This report demonstrates that beyond those initial measures of proliferation and expansion that substrate stiffness modulates iTreg suppressive capacity and epigenetic programming.

[0072] By optimizing the activating substrate stiffness, we were able to not only outperform classic plate-bound control but also perform as well as the clinical gold standard in Treg production with a simple planar surface. The stiffest PDMS substrate was able to induce more tTreg-like epigenetic profiles and more suppressive capacity than both plate-bound controls and Dynabeads. They are also appropriate for the ex vivo production of Tregs despite their lack of physiological relevance.

[0073] Suppressive capacity is one major parameter for the efficacy of Treg adoptive therapy. In our study, a 1 :1 ratio of Tregs to Tconvs exhibited strong suppressive capabilities. Treg suppressive capacities decrease as the ratios of Tregs to Tconvs decrease: 1 :4 ratio or lower showed weak to low suppressive response. This is in line with other studies that show 1 :1 or 1 :2 ratios of Tregs to Tconvs being needed to exert the immune-suppressive response in vivo (49, 50). Most tumormicroenvironments have been reported to be stiffer than normal tissue (51-53). These reports agree with our findings that the stiffer surface induces more suppressive Tregs. Overall, these studies signify the impact of mechanical cues on cellular functions and the importance of substrate stiffness optimization for the production of all therapeutic products.

[0074] Epigenetic modifications are important for normal growth and development in response to environmental inputs, including mechanical stiffness. Killaars et al. have discovered that substrate stiffness induced different chromatin remodeling in human mesenchymal stem cells (39). A more remarkable finding is that short-term culture on the stiff substrate results in reversible mechanical memory, whereas the extending mechanical dose leads to irreversible mechanical memory. Epigenetic modification could be a way for cells to remember mechanical cues and influence long-term cell fate. The idea of mechanical memory could explain the difference in Treg induction persisting across stiffness and even after restimulation. On the other hand, Zhao et al. demonstrated that extracellular matrix stiffness regulates DNA methylation in mouse embryonic stem cells and mouse embryonic fibroblast by translocating DNMT3L in a PKC pathway-dependent manner (38). These results are in line with our findings that substrate stiffness can regulate epigenetic profiles, and our belief that epigenetic modification is an important parameter worth investigating in cell biology.

[0075] Several studies also investigated how TOR signaling regulates epigenetic profiles in T cells but not in the context of mechanosensing. Ohkura et al. discovered that T cell receptor stimulation induced epigenetic changes in multiple essential Treg functional genes (54). Wakamatsu et al. have shown that the Foxp3 CNS2 of induced Tregs is partially demethylated depending on the strength of TCR stimulation (55). This report establishes a mechanosensing response on DNA methylation levels in the process of human Treg induction. Thus, the stability of Tregs could be affected by tissue-specific environmental factors, including the stiffness of the extracellular environment.

[0076] Treg development requires both TCR-induced epigenetic changes on Treg specific regions and FOXP3 expression. CpG hypomethylation in Treg-related genes, such as CTLA4 exon2, FOXP3 intronl , TNFRSF18 exon 5, IKZF4 intron 11b, and IL2RA intron 1a, is specifically limited to Tregs and persists after stimulation (54,56). The formation of Treg-specific DNA hypomethylation is FOXP3 independent, as the expression of Treg-related genes has already begun prior to FOXP3 expression during Treg development in the thymus (57). In addition, DNA hypomethylation and histone modification are still present in most of the Treg signature genes that are upregulated in FOXP3-deficient cells. These epigenetic profiles contribute to Treg suppressive activity and lineage stability (58), and changes in methylation patterns can lead to imbalanced immune responses.

[0077] Many challenges still need to be addressed to unleash the full potential of Treg adoptive therapy in treating autoimmune diseases. This process could start with optimizing the mechanical environment for therapeutic cell production. A 1 :25 (870kPa) formula of planar PDMS surface is the ideal stiffness for Treg induction since it generates as many cells as 1 :59 (17kPa) and is as functional as 1 :10 (2600kPa). In addition, it also outperforms the clinical gold standard, Dynabeads, in suppressive capacity, generating the same amount of cells.

[0078] It is noted that additional factors impact the mechanosensing response. In particular, earlier studies using polyacrylamide gels showed increasing induction with greater substrate stiffness, an apparently contradictory result (35). However, the range of elastic modulus examined in those studies (7.5 - 140 kPa) was much lower than used in this report. It is possible that Treg induction as measured by FOXP3 expression, like other functions of conventional T cells including activating and spreading (48, 59), is biphasic with respect to mechanical stiffness and the two reports capture opposite sides of a curve bracketing an optimal response. In addition, T cell mechanosensing is dependent on the concentration of activating ligand (48); identification of differences in ligand concentration or substrate chemistry could also reconcile the results observed on polyacrylamide and PDMS, and serve to guide future biomaterial design.

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[0080] All documents cited in this application are hereby incorporated by reference as if recited in full herein. In the event of a conflict between the teachings of this application and those of the incorporated documents, the teachings of this application control.

[0081] Although illustrative embodiments of the present disclosure have been described herein, it should be understood that the disclosure is not limited to thosedescribed, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the disclosure.

Claims

What is Claimed is:

1. A substrate for optimizing iTreg induction and suppressive activity having a Young’s Modulus in the range from 15 kPa to 3000 kPa.

2. The substrate of claim 1 , having a Young’s Modulus of 870 kPa.

3. A process for preparing a substrate, comprising the steps of:(a) mixing an elastomer with a curing agent at a desired ratio;(b) degassing and curing the mixture; and(c) coating the cured mixture with at least one antibody.

4. The process of claim 3, comprising attaching the cured mixture to at least one antibody in step (c).

5. The process of claim 3, wherein the elastomer is selected from the group consisting of aliphatic polyesters, polyacrylamide, polyhydroxyalkanoates, polyurethanes, polyalkylene oxides, siloxanes, polyvinylalcohol, polyvinylpyrrolidone, polylysine, collagen, gelatin, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides, polysaccharides, and combinations thereof.

6. The process of claim 5, wherein the siloxanes can be cyclic or linear.

7. The process of claim 3, wherein the elastomer is hydrophobic.

8. The process of claim 3, wherein the elastomer is polydimethylsiloxane (PDMS).

9. The process of claim 3, wherein the curing agent is a cross-linking agent selected from the group consisting of pentaerythritol, (3- Aminopropyl)triethoxysilane (APTES), (3-Aminopropyl)trimethoxysilane, 3- Aminopropyl-methyl-diethoxysilane, 3-(Dimethoxymethylsilyl)propylamine, tetraacetoxysilane, triacetoxy methylsilane; triactetoxy ethylsilane, tetraethyl silicate, and combinations thereof.

10. The process of claim 3, wherein the desired ratio is a weight ratio of the curing agent and the elastomer in the range from 1 : 10 to 1 :60.

11. A substrate made by the process of claim 3.

12. A method for producing iTregs in clinically useful quantities and with clinically relevant suppressive activity comprising the steps of:(a) producing a substrate by the process of claim 3;(b) culturing regulatory T cells with an induction media on the substrate produced; and(c) collecting the induced cells after a sufficient period of time and repeating step (b).

13. The method of claim 12, comprising collecting the induced cells and repeating step (b) every 3 days for up to 15 days.

14. The method of claim 12, wherein the substrate produced in step (a) has a Young’s Modulus in the range from 15 kPa to 3000 kPa.

15. The method of claim 12, wherein the substrate produced in step (a) has a Young’s Modulus of 870 kPa.

16. The method of claim 12, wherein the induction media is selected from the group consisting of TGF-p, retinoic acid, rapamycin, IL-2, or combinations thereof.

17. A method for treating or ameliorating the effects of a disease in a subject in need thereof, comprising administering to the subject an effective amount of iTregs made by the method of claim 12.

18. The method of claim 17, wherein the subject is a human.

19. The method of claim 17, wherein the disease is selected from the group consisting of an autoimmune disease, an inflammatory complication, and a graft-versus-host disease (GvHD).

20. The method of claim 17, wherein the disease is selected from the group consisting of chronic GvHD, hyperinflammatory COVID, Type 1 Diabetes, and Systemic Lupus Erythematosus.

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