Artificial antigen presenting cells to improve t cell function

Abstract

Artificial antigen presenting cells (aAPCs) improve immune cell response. In particular, these aAPCs are constructed from antioxidant biomaterials and used in the treatment of, for example, cancer, infections, autoimmunity.

Description

DOCKET: 348358.17302 ARTIFICIAL ANTIGEN PRESENTING CELLS TO IMPROVE T CELL FUNCTION The present application claims the benefit of U.S. provisional application 63 / 538,770 filed September 15, 2023, which is incorporated by reference herein in its entirety. FIELD

[0001] Artificial antigen presenting cells (aAPCs) for inducing T cell responses in vitro or in vivo. BACKGROUND

[0002] Adoptive T cell therapy (ACT), a cancer immunotherapy, relies on autologous CD8+ T cells expanded ex vivo. This treatment has led to complete remission and durable complete responses in melanoma patients. However, issues with T cell potency reduce the feasibility of ACT. CD8+T cell phenotype is one indicator of potency; studies have shown that central memory CD8+T are more efficacious due to increased stemness and ability to persist in vivo. There are critical parameters that affect CD8+T cells during ex vivo expansion, such as signal intensity, nutrient availability, and overall time of culture. SUMMARY

[0003] We have now constructed artificial antigen presenting cells (aAPCs) using antioxidant biomaterials which modulate an immune response.

[0004] In one aspect, an artificial antigen presenting cell (aAPC) aAPC is composed of an antioxidant biomaterial. In certain embodiments, the antioxidant biomaterial is biological comprising biomaterial derived or obtained from plants, animals, algae, fungi, bacteria, synthetic materials or combinations thereof. In certain embodiments, the aAPC is a nanoparticle. In certain embodiments, the diameter of the nanoparticle is in a range from about 200 nm to about 1000 nm. In certain embodiments, the diameter of the nanoparticle is in a range from about 250 nm to about 900 nm. In certain embodiments, the diameter of the nanoparticle is in a range from about 400nm to about 800nm.16265565

[0005] In certain embodiments, the antioxidant biomaterial is derived from plants, animals, algae, fungi, bacteria, synthetic materials or combinations thereof. In certain embodiments, the antioxidant biomaterial is derived from algae in combination with one or more antioxidant biomaterials derived from plants, animals, fungi, bacteria or synthetic materials. In certain embodiments, the antioxidant biomaterial is derived from algae. In certain embodiments, the antioxidant biomaterial derived from algae comprises alginate, agarose, carrageenan, fucoidan or combinations thereof. In certain embodiments, the antioxidant biomaterial derived from algae comprises alginate. In certain embodiments, the antioxidant biomaterial modulates reactive oxygen species (ROS) and oxidative stress in vitro or in vivo.

[0006] In certain embodiments, the aAPC, further comprises an antigenic peptide, a major histocompatibility complex (MHC) molecule, a T cell specific ligand, a T cell activation signal, T cell costimulatory signal, or combinations, thereof expressed on a surface of the nanoparticle. In certain embodiments, the T cell activation signal comprises a polypeptide comprising a major histocompatibility complex Class I (MHC class I) molecule loaded with an antigen, an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD2 antibody, antibody fragments or combinations thereof.

[0007] In another aspect, the present disclosure provides an artificial antigen presenting cell (aAPC) engineered to activate T cells, the aAPC comprising an exogenous antigen presenting poly peptide and an exogenous antigen-presenting polypeptide on the cell surface, wherein the exogenous antigen-presenting polypeptide is an MHC class I single-chain fusion protein or an MHC class II single-chain fusion protein.

[0008] In some aspects, an artificial antigen presenting cell (aAPC) engineered to activate T cells, comprises a dendritic cell phenotype. In certain aspects, a dendritic cell phenotype comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDC1), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.

[0009] In certain aspects, the aAPC comprises one or more dendritic cell markers. In certain embodiments, the dendritic cell markers comprise BDCA-1, CD8, CD8alpha, CD11b, 2 162665565.1CD11c, CD103, CD205, MHC Class II, BDCA-2, BDCA-4, CD11clowCD45RA, CD123 ILT-7, MHC Class IIlowTLR7, TLR9 or combinations thereof.

[0010] In some aspects, an artificial antigen presenting cell (aAPC) engineered to activate T cells, comprises a macrophage cell phenotype.

[0011] In certain aspects, the artificial antigen presenting cell (aAPC) comprises one or more macrophage markers. In certain embodiments, the macrophage markers comprise M1 macrophage markers, M2 macrophage markers or combinations thereof. In certain embodiments, the M1 macrophage markers comprise CD86, CD80, CD68, MHC class II, IL-1R, TLR2, TLR4, iNOS, SOCS or combinations thereof. In certain embodiments, the M2 macrophage markers comprise CD163, MHC class II, SR, MMR / CD206, CD200R, TGM2, DecoyR, IL-1R II, CD86, CD163, TLR1, TLR8, VEGF or combinations thereof.

[0012] In certain embodiments, the MHC class I single chain fusion protein comprises an α chain and a β2m chain. In certain embodiments, the MHC class I single chain fusion protein further comprises a membrane anchor. In certain embodiments, the foreign antigen polypeptide is connected to the MHC I single chain fusion protein via a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the MHC class II single chain fusion protein comprises an α chain and a β chain. In certain embodiments, the MHC class II single chain fusion protein further comprises a membrane anchor. In certain embodiments, the foreign antigen polypeptide is connected to the MHC II single chain fusion protein via a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the anchor comprises glycophorin A (GPA) protein or comprises the transmembrane domain of small integral membrane protein 1 (SMIM1). In certain embodiments, the foreign antigen polypeptide is covalently linked to the foreign antigen-presenting polypeptide. In certain embodiments, the foreign antigen polypeptide is non-covalently bound to the foreign antigen-presenting polypeptide.

[0013] In certain embodiments, the aAPC further comprises at least one exogenous co- stimulatory polypeptide on the cell surface. In certain embodiments, the at least one exogenous co-stimulatory polypeptide is 4-1BBL, LIGHT, anti-CD28, CD80, CD86, CD70, OX40L, GITRL, TIM4, SLAM, CD48, CD58, CD83, CD155, CD112, IL-15Rα fused to IL-15, IL-21, ICAM-1, ligand of LFA-1, anti-CD3, and combinations thereof. In certain embodiments, the 3 162665565.1aAPC comprises at least 2, at least 3, at least 4, or at least 5 exogenous co-stimulatory polypeptides on the cell surface.

[0014] In certain embodiments, the aAPC further comprises an exogenous cytokine polypeptide on the cell surface. In certain embodiments, the exogenous cytokine polypeptide is IL-2, IL-15, 15Rα fused to IL-15, IL-7, IL-12, IL-18, IL-21, IL-4, IL-6, IL-23, IL-27, IL-17, IL- 10, TGF-β, IFNγ, IL-1β, GM-CSF, and IL-25.

[0015] In certain embodiments, aAPCs are capable of activating T cells comprising CD8+T cells, CD4+T cells, stimulation of T cell cytotoxic activity, stimulation of cytokine secretion by T cells, and / or any combination thereof.

[0016] In certain embodiments, the aAPC regulate regulatory T cell (Treg) responses.

[0017] In certain embodiments, the aAPC comprises an inhibitory polypeptide. In some embodiments, the inhibitory polypeptide is IL-35, IL-10, VSIG-3, PD-L1 or a LAG3 agonist.

[0018] In certain embodiments, the aAPC further comprises encapsulating a peptide, a nucleic acid, a cytokine, an adjuvant, a therapeutic agent, a toxin, or combinations thereof.

[0019] In another aspect, a pharmaceutical composition comprises an artificial antigen presenting cell (aAPC) embodied herein.

[0020] In another aspect, a method of treating cancer comprises administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) embodied herein.

[0021] In another aspect, a method of treating an infectious disease comprises administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) embodied herein.

[0022] In another aspect, a method of modulating an immune response comprises administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) embodied herein.

[0023] In another aspect, a method of treating an autoimmune disease comprising administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) embodied herein.

[0024] In another aspect, an artificial antigen presenting cell (aAPC) is formulated as a active material.

[0025] Definitions 4 162665565.1

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, cell culture, molecular genetics, and biochemistry).

[0027] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0028] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0029] As used herein, by “administering” is meant a method of giving a dosage of a composition described herein (e.g., an aAPC and / or a pharmaceutical composition thereof) to a subject. The compositions utilized in the methods described herein can be administered by any suitable route, including, for example, by inhalation, nebulization, aerosolization, intranasally, intratracheally, intrabronchially, orally, parenterally (e.g., intravenously, subcutaneously, or intramuscularly), orally, nasally, rectally, topically, or buccally. In some embodiments, a composition described herein is administered in aerosolized particles intratracheally and / or intrabronchially using an atomizer sprayer (e.g., with a MADgic® laryngo-tracheal mucosal atomization device). 5 162665565.1

[0030] As used herein, the terms “agent” or “moiety” are meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, hormones, oligonucleotides, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

[0031] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0032] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0033] In the descriptions herein and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in 6 162665565.1the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0034] As used herein, the term “cellular immune response” can be used interchangeably with the term “cell-mediated immune response” and refers to an immune response that does not predominantly involve antibodies. Instead, a cellular immune response involves the activation of different immune cells (e.g., phagocytes and antigen-specific cytotoxic T-lymphocytes) that produce various effector molecules (e.g., cytokines, perforin, granzymes) upon activation (e.g., via antigen stimulation). As used herein, the term “humoral immune response” refers to an immune response predominantly mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. The term “antibody-mediated immune response” refers to an aspect of a humoral immune response that is mediated by antibodies.

[0035] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, equivalents thereof--and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0036] An “immune response,” as used herein, refers to a biological response within a vertebrate against foreign agents, e.g., virus, or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell. 7 162665565.1Accordingly, an immune response can comprise a humoral immune response (e.g., mediated by B- cells), cellular immune response (e.g., mediated by T cells), or both humoral and cellular immune responses. In some aspects, an immune response is an “inhibitory” immune response. An “inhibitory” immune response is an immune response that blocks or diminishes the effects of a stimulus (e.g., antigen). In certain aspects, the inhibitory immune response comprises the production of inhibitory antibodies against the stimulus. In some aspects, an immune response is a “stimulatory” immune response. A “stimulatory” immune response is an immune response that results in the generation of effectors cells (e.g., cytotoxic T lymphocytes) that can destroy and clear a target antigen (e.g., tumor antigen or viruses).

[0037] As used herein, the term “immune cells” refers to any cells of the immune system that are involved in mediating an immune response. Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof. In some aspects, an immune cell expresses CD3. In certain aspects, the CD3-expressing immune cells are T cells (e.g., CD4+T cells or CD8+T cells). In some aspects, an immune cell that can be targeted with a targeting moiety (e.g., anti-CD3) comprises a naive CD4+T cell. In some aspects, an immune cell comprises a memory CD4+T cell. In some aspects, an immune cell comprises an effector CD4+T cell. In some aspects, an immune cell comprises a naïve CD8+T cell. In some aspects, an immune cell comprises a memory CD8+T cell. In some aspects, an immune cell comprises an effector CD8+T cell. In some aspects, an immune cell is a dendritic cell. In certain aspects, a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic cell 1 (cDC1), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme- expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.

[0038] As used herein, the term “modulate” includes to “increase” or “decrease” one or more quantifiable parameters, optionally by a defined and / or statistically significant amount. By “increase” or “increasing,” “enhance” or “enhancing,” or “stimulate” or “stimulating,” refers generally to the ability of one or more aAPC compositions in accordance with the present disclosure to produce or cause a greater physiological response (i.e., downstream effects) in a cell or in a subject relative to the response caused by either no aAPC composition or a control compound. Relevant physiological or cellular responses (in vivo or in vitro) upon administration of aAPC 8 162665565.1compositions will be apparent to persons skilled in the art. An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e.g., 500, 1000 times), including all integers and decimal points in between and above 1 (e.g., 1.5, 1.6, 1.7.1.8), the amount produced by no aAPC composition or a control compound. The term “reduce” or “inhibit” may relate generally to the ability of one or more aAPC compositions to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of a disease, illness or condition, such as cancer, inflammation or pain. A “decrease” in a response may be “statistically significant” as compared to the response produced by no aAPC composition or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0039] As used herein, the term “T cell” or “T-cell” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T-cells include all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T-cells, T-regulatory cells (Treg), and gamma-delta T cells. Among the sub-types and subpopulations of T cells and / or of CD4+and / or of CD8+T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCMXcentral memory T (TCMeffector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0040] A “naive” T cell refers to a mature T cell that remains immunologically undifferentiated (i.e., not activated). Following positive and negative selection in the thymus, T 9 162665565.1cells emerge as eitherCD4+ or CD8+naive T cells. In their naive state, T cells express L-selectin (CD62L+), IL-7 receptor-α (IL-7R-α), and CD132, but they do not express CD25, CD44, CD69, or CD45RO. As used herein, “immature” can also refer to a T cell which exhibits a phenotype characteristic of either a naive T cell or an immature T cell, such as a TSCM cell or a TCM cell. For example, an immature T cell can express one or more of L-selectin (CD62L+), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, and LFA-1. Naive or immature T cells can be contrasted with terminal differentiated effector T cells, such as TEMcells and TEFFcells.

[0041] As used herein, the term “effector” T cells or “TEFF” cells refers to a T cell that can mediate the removal of a pathogen or cell without requiring further differentiation. Thus, effector T cells are distinguished from naive T cells and memory T cells, and these cells often have to differentiate and proliferate before becoming effector cells.

[0042] As used herein, the term “memory” T cells refer to a subset of T cells that have previously encountered and responded to their cognate antigen. In some aspects, the term is synonymous with “antigen-experienced” T cells. In some aspects, memory T cells can be effector memory T cells or central memory T cells. In some aspects, the memory T cells are tissue-resident memory T cells. As used herein, the term “tissue-resident memory T cells” or “TRM cells” refers to a lineage of T cells that occupies tissues (e.g., skin, lung, gastrointestinal tract) without recirculating. TRM cells are transcriptionally, phenotypically and functionally distinct from central memory and effector memory T cells which recirculate between blood, the T cell zones of secondary lymphoid organs, lymph and nonlymphoid tissues. One of the roles of TRM cells is to provide immune protection against infection in extra lymphoid tissues.

[0043] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0044] The terms “treatment”, or “treating” as used herein, is defined as the application or administration of an aAPC composition and / or combination of therapeutic agents (e.g., an anti- inflammatory agent, a cancer preventing and / or therapeutic agent (e.g., chemotherapeutic)) to a patient, or application or administration of an aAPC composition to an isolated tissue or cell line 10 162665565.1from a patient, who has a disorder with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, or symptoms of the disease or disorder. The term “treatment” or “treating” is also used herein in the context of administering agents, e.g., an aAPC composition prophylactically. The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0045] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Concentrations, amounts, cell counts, percentages and other numerical values may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0046] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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.

[0049] FIGS.1A-1H show the fabrication and characterization of alginate based nanoparticles and and impact on B6 CD8+ T cell ROS production. FIG.1A: Schematic of alginate nanoparticle production. FIG.1B: TEM image depicting the size and shape of the alginate nanoparticles. FIGS.1C-D) Dynamic light scattering data showing z-average, particle size distribution, and zeta potential with standard deviation. FIG.1E: Schematic of experimental 11 162665565.1setup for ROS measurements. FIG.1F: Flow data depicting MitoSOX staining for mitochondrial ROS. FIG.1G: Percentage of cells positive for MitoSOX green and H) corresponding MFI (n = 4, one-way ANOVA with Tukey’s multiple comparison). *p < 0.033, **p < 0.002, ***p < 0.001..

[0050] FIGS.2A-2E demonstrate that bare alginate particles help reduce cellular stress and are compatible with CD8+T cell expansion. CD8+T cells after 48 hour incubation with anti- CD3 and anti-CD28. FIG.2A) Schematic of experiment setup. Total percentage of FIG.2B) total oxidative stress and FIG.2C) superoxide and MFI of cells positive for FIG.2D) oxidative stress and FIG.2E) superoxide from flow data. (*p < 0.033, **p < 0.002, ***p < 0.001; n = 4; paired t- test.

[0051] FIGS.3A-3E demonstrate the in vitro expansion of CD8+T cells with Alginate nanoparticles FIG.3A) Experiment layout FIG.3B) CFSE data on day 3 after incubation with anti-CD3 and anti-CD28. FIG.3C) Proliferation breakdown by generation based on CFSE histogram peaks. FIG.3D) LAG3+PD1+TIM3+and FIG.3E) Percentage of CD44+on day 7 after harvest. (*p < 0.033, **p < 0.002, ***p < 0.001; n = 3; Tukey multiple comparison).

[0052] FIGS.4A-4G demonstrate that a base composition of aAPC results in differences in memory related transcription factors and persistence. FIG.4A) Schematic of aAPCs. FIG.4B) CFSE on day 3. FIG.4C) Fold expansion. Percentage of FIG.4D) TCF+, FIG.4E) Eomes+, FIG. 4F) Tbet+T cells on day 7.2C CD8+T cells were activated with alginate or iron dextran aAPCs for 7 days and transferred to B6 mice. Blood was analyzed on day 5, 8, 14, and 21 for FIG.4G) percentage of transferred cells in blood. (*p < 0.033, **p < 0.002, ***p < 0.001; n = 3; One- way ANOVA).

[0053] FIGS.5A-5E demonstrate the improvement of CD8+T cell phenotype, persistence, and killing with Alginate based aAPC. FIG.5A) Flow data of CD8+T cells on day 7 gated on CD44 and CD62L. FIG.5B) Percentage of CD44+CD62L+CD8+T cells on day 7. FIG.5C) Schematic of workflow for an in vivo killing assay. Percent killing on days 7 and 21 post treatment injection in the FIG.5D) spleen and FIG.5E) lymph nodes. (*p < 0.033, **p < 0.002, ***p < 0.001; n = 5; Tukey multiple comparison).

[0054] FIGS. 6A-6E demonstrate that alginate aAPC activated CD8+T cells significantly reduce tumor burden. FIG. 6A) no treatment, FIG. 6B) naïve, and FIG. 6C) ex vivo activated 12 162665565.1treatment groups separately and FIG. 6D) combined. FIG. 6E) The Kaplan survival curve. (*p < 0.033, **p < 0.002, ***p < 0.001; n = 4-5; Sidak's multiple comparisons test, SEM depicted).

[0055] FIG.7A-7F: Alginate-based aAPCs expand 2C transgenic and B6 CD8+ T cells in an antigen-specific manner. A) Schematic of alginate-based aAPCs. B) The average total protein conjugated and the C) histograms showing proliferation by CFSE. D) The fold expansion of the 2C transgenic CD8+ T cells on day 7 (n = 3-6, one-way ANOVA with Tukey’s multiple comparison). E) Flow cytometry data showing KbSIY+ CD8+ B6 T cells on day 7. F) Total KbSIY+CD8+ B6 T cell numbers on day 7 (n = 3, Student’s t-test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0056] FIG.8A-8E: CD8+ T cells expanded with alginate-based aAPCs demonstrate high target cell killing. A) Schematic of workflow for the in vivo killing assay. Flow data depicting CFSEhiand CFSElowpopulations in the B) spleen and D) lymph nodes on days 7 and 21 post- treatment injections. Percent killing on days 7 and 21 in the C) spleen and E) lymph nodes (n = 3- 5; one-way ANOVA with Tukey’s multiple comparison). *p < 0.033, **p < 0.002, ***p < 0.001.

[0057] FIG.9A-9D: CD8+ T cells expanded with alginate-based aAPCs show therapeutic efficacy in an adoptive transfer tumor model. A) Schematic of workflow for tumor treatment model. B) Individual tumor growth curves for no treatment, naïve, and alginate-based aAPC expanded treatment groups. C) Combined tumor growth and D) Kaplan-Meier survival curve (n = 5; two way ANOVA with Sidak's multiple comparisons test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0058] FIG 10A-10K: CD8+ T cells expanded with alginate-based aAPCs exhibit memory- like qualities and show improved persistence in vivo. Flow data depicting 2C CD8+ T cells gated for A) TCF1, C) Eomes, E) Tbet, and G) IL-2. Percent positive 2C CD8+ T cells for B) TCF1, D) Eomes, F) Tbet (n = 3, Student’s t-test), and H) IL-2 (n = 5, Student’s t-test). I) Schematic for in vivo persistence experimental set up. J) Percentage of adoptively transferred 2C CD8+ T cells on day 5, 8, 14, and 21 post-transfer. K) Percentage of adoptively transferred 2C CD8+ T cells in the spleen and lymph nodes on day 21 (n = 5, one way ANOVA). *p < 0.033, **p < 0.002, ***p < 0.001.

[0059] FIG. 11A-11D. Adoptively transferred cells expanded with alginate-based aAPCs significantly slow tumor growth and promote host survival. A) Schematic of workflow for preventative tumor treatment model. Individual tumor growth curves for A) no treatment, iron oxide aAPC, and alginate-based aAPC expanded treatment groups. C) Combined tumor growth and D) 13 162665565.1survival curve (n = 5, two way ANOVA wtih Sidak's multiple comparisons test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0060] FIG.12A-12B Day 7 activation markers for B6 CD8+ T cells expanded with alginate nanoparticles. A) Percentage of positive population for PD-1, Tim-3, and LAG-3 or B) CD44 (n = 4, Student’s t-test).

[0061] FIG.13A-13C: Protein conjugation to alginate nanoparticles. A) Conjugated protein for both anti-CD3 and anti-CD28. (n=9) B) Total protein on day 0 and day 7 (n = 4). C) Percentage of protein bound on day 7 (n = 4).

[0062] FIG. 14A-14B: Antigen specific expansion of B6 CD8+ T cells. A) Total cell number and B) percentage of KbSIY+ CD8+ T cells (n = 3, Student’s t-test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0063] FIG. 15: Tumor growth curve. Tumor growth for naïve 2C CD8+ T cell and alginate-based aAPC expanded 2C CD8+ T cell treated groups (n = 5, two way ANOVA with Sidak’s multiple comparison test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0064] FIG. 16A-16B: Cytokine production from aAPC activated 2C CD8+ T cells. A) Percentage of cells positive for 0, 1, or 2 or more cytokines (n=3, two way ANOVA with Sidak’s multiple comparison test). B) Percentage of cells positive for TNF-alpha, INF-gamma, or granzyme B (n=3, Student’s t-test). *p < 0.033, **p < 0.002, ***p < 0.001.

[0065] FIG.17A-17B: In vivo persistence of aAPC activated 2C CD8+ T cells. A) Trend lines for percentage of transferred 2C cells over time. B) Percentage of CD3+ CD8+ T cells in the spleen and lymph nodes on day 21 (n=5, Student’s t-test).

[0066] FIG.18A-18B: Phenotypic analysis of alginate-based aAPC expanded 2C CD8+ T cells. A) Flow plot and B) chart depicting central memory like (CD44+CD62L+) T cells (n=3). DETAILED DESCRIPTION

[0067] In CD8+T cells, reactive oxygen species (ROS) and oxidative stress accumulation, caused by activation, activates FOXO1 to transcribes antioxidants to reduce ROS which improve memory formation. This suggests that oxidative stress reduction may be linked to proper memory T cell formation. Thus, to preferentially expand central memory CD8+T cells, a newly synthesized artificial antigen presenting cells (aAPCs) was developed from alginate, an antioxidant biomaterial, to allow for greater control of CD8+T cell phenotype and efficacy. 14 162665565.1

[0068] Antioxidant Biomaterials

[0069] The accumulation of ROS is responsible for the lipid peroxidation of the hydroxyl radical, hydrogen and superoxide anion. ROS are associated with alterations and damage to cellular constituents (membrane lipids, nucleic acids and proteins), which signal several complications for human health (inflammation, proliferation and differentiation of stem cells, chronic wounds, bone defects, Parkinson’s and Alzheimer’s diseases, among others) including memory T cell formation.

[0070] Antioxidant biomaterials in the formulation of aAPCs can come from a variety of sources including synthetic or natural renewable sources.

[0071] Natural biomaterials can come from living organisms such as animals, plants, fungi, algae, and bacteria. Thus, there is a diversity of biomaterials spread throughout nature, which can be formed by chains of already known molecules such as proteins, carbohydrates, and lipids.

[0072] Proteins are produced especially by members of the animal kingdom; collagen, keratin, and gelatin, in particular, are used for pharmaceutical and food applications. Collagen and keratin are long filamentous fibrillar proteins that make up the extracellular matrix of animal cells, known as scleroproteins. The versatility of these biomaterials is reflected in diverse areas of application in biomedicine, such as wound healing, tissue engineering, surface coating of medical devices, and skin supplementation. In this sense, the biocompatibility of these proteins contributes to the successful application of these biomaterials. In addition, gelatin can be used as an encapsulating agent, as a delivery system for drugs and bioactive compounds, and in the production of biological tissues, due to its biocompatibility, non-toxicity, and biodegradability. Fatty acids and waxes are produced by insects such as bees. These components can be used to develop solid lipid nanoparticles used as drug delivery systems for different medications.

[0073] The biomaterials derived from carbohydrates are chiefly presented as polysaccharides, which plants, bacteria, and algae can originate. Cellulose, consisting of a chain of glucose joined by β-(1→4) bonds, can be produced by plants and bacteria. The use of cellulose as a biomaterial involves the production of antimicrobial, biodegradable, and antioxidant packaging, emulsifier, texture modifier, drug and polyphenol delivery systems, skin care products, and heart valves and blood tubes. Starch, a chain of glucose residues linked together by 15 162665565.1α-(1→4) glycosidic bonds (amylose) and ramified by α-(1→6) glycosidic bonds (amylopectin), is formed as an energy reserve in plants. In the case of biomaterials, starches can be used in the production of biopolymers such as ethylene and polyethylene. Chitin and chitosan, widely used in tissue engineering due to their biocompatibility, can be produced by certain fungi and crustaceans. Chitin is a semicrystalline homopolymer of β-(1→4)-linked N-acetyl-D-glucosamine. Chitosan is obtained by boiling chitin in potassium hydroxide, making it soluble in organic acids. Gelling polysaccharides such as alginate, agarose, and carrageenan are mainly extracted from algae.

[0074] Among the biomaterials that have been developed, antioxidant can inhibit excess superoxide, hydroxyl, hydroperoxyl, and hydrogen peroxide radicals, also known as reactive oxygen species (ROS), which characterizes their antioxidant capacity. ROS are essential for the regulation of metabolism. However, their excess, caused by several extrinsic factors, can affect their healthy functioning. As provided herein, use of an antioxidant biomaterial, such as alginate, has an effect on the immune cell function, in particular T cell responses. For a review on antioxidant biomaterials, see, for example Pedro AC, Paniz OG, Fernandes IAA, et al. The Importance of Antioxidant Biomaterials in Human Health and Technological Innovation: A Review. Antioxidants (Basel).2022;11(9):1644. Published 2022 Aug 24. doi:10.3390 / antiox11091644.

[0075] Artificial Antigen Presenting Cells (aAPCs)

[0076] Active immune responses depend on efficient presentation of antigens and co- stimulatory signals by antigen-presenting cells (APCs). APCs are part of the innate immune system and act as an interface between antigen recognition and the adaptive response of T cells during antigen presentation. Activation of T cells requires the appropriate activation and integration of three signals. The first signal is the antigen, which is presented in the context of the major histocompatibility complex, and its recognition by the T cell receptor (TCR). The first signal is not sufficient to activate T cells. Activation continues with a second signal that involves the participation of surface molecules expressed on dendritic cells that interact with their respective receptors on the T cell. The third signal involves the production of cytokines, which not only favor the activation state but also promote the polarization of T cells into their various helper / cytotoxic subpopulations. In that dynamic microenvironment, the spatiotemporal 16 162665565.1expression of various co-stimulatory molecules on dendritic cells and T cells, as part of the second signal, is the key to regulating T cell activation, inhibition, survival, and polarization.

[0077] Artificial APCs (aAPCs) are engineered platforms for T cell activation and expansion which mimick the interaction between DCs and T cells. They include multiple systems, including synthetic biomaterials that have been engineered to activate and / or expand desirable immune cell populations (e.g., T cells). These systems may act by mimicking the interaction between DCs and T cells. For instance, several cell-sized, rigid, beads, such as latex microbeads, polystyrene-coated magnetic microbeads and biodegradable poly(lactic-co-glycolic acid) microparticles, have been developed. The efficacy of these beads in inducing activation and / or expansion of immune cells appears to be highly dependent on the properties of the materials used. For example, beads greater than 200 nm are typically retained at the site of inoculation, while smaller particles may be taken up by DCs (see, e.g., Reddy et al. (2006) J. Control. Release 112: 26– 34).

[0078] Similar to APCs, the aAPCs embodied herein further comprise stimulatory and costimulatory molecules. Co-stimulatory molecules are transmembrane glycoproteins that induce activation or inhibition cascades that enhance or diminish TCR signaling. Stimulatory, or activating signals (co-stimulation by CD28 or CD40), lead to the production of growth factors, cell expansion, and survival. Inhibitory signals (co-inhibition by PD1 or CTL-4) attenuate TCR- induced signals, resulting in decreased cell activation, inhibition of growth factor production, inhibition of cell cycle progression, and in some cases, promotion of cell death.

[0079] Families of co-stimulatory molecules: Co-stimulatory molecules are divided into two main families by their molecular structure. The first (Table 1) is the immunoglobulin superfamily which includes CD226, the CD2 / signaling lymphocytic activation molecule family, T cell immunoglobulin and mucin (TIM) family, butyrophilin (BTN) family, and leukocyte- associated immunoglobulin-like receptor (LAIR) family. Because of its historical relevance, the most studied is the B7 family, which includes CD80, CD86, and its receptor CD28. The second (Table 8) is the tumor necrosis factor superfamily (TNFR SF), which includes three subfamilies, the divergent type (OX-40, CD27, glucocorticoid-induced TNFR-related protein), the S-type (CD267), and the conventional type [FAS, herpes virus entry mediator, receptor activator of 17 162665565.1nuclear factor kappa-B (RANK), and CD40]. CD40 and its ligand CD40L are the most investigated co-stimulation molecules of the TNFR SF.

[0080] Table 1 Immunoglobulin super family co-stimulatory molecules IgSF co-stimulatory Function Cells expressing the Ligand Cells expressing the molecules receptor ligand CD28 Activation Constitutive in T cells CD80, CD86 CD80: Inducible in dendritic cells, monocytes, B and T cells. CD86: Constitutive in dendritic cells, monocytes, B and T cells ICOS (CD278) Activation Inducible in T, B, and ICOSL Constitutive in NK cells macrophages, dendritic cells, B and T cells CTLA-4 (CD152) Inhibition Inducible in T cells CD80, CD86 CD80: Inducible in dendritic cells, monocytes, B and T cells. CD86: Constitutive in dendritic cells, monocytes, B and T cells PD-1 (CD279) Inhibition Inducible in T, and B PD-L1, PD- PD-L1: Constitutive in cells, macrophages L2 dendritic cells, B and T cells. PD-L2: Inducible in dendritic cells and monocytes PD-1H (VISTA) Inhibition Monocytes, Unknown Unknown neutrophils, T cells BTLA (CD272) Inhibition B and T cells HVEM, Monocytes, B and T cells UL144 18 162665565.1B71 (CD80), B72 (CD86) Activation / Inhi CD80: Inducible in CD28, CD28: Constitutive in T bition dendritic cells, CTLA-4 cells. CTLA-4: Inducible in monocytes, B and T T cells cells. CD86: Constitutive in dendritic cells, monocytes, B and T cells B7H1 (CD274, PDL1) Inhibition Constitutive in PD-1, B71 PD-1: Inducible in dendritic cells, macrophages, B and T cells. monocytes, B and T CD80: Inducible in cells dendritic cells, monocytes, B and T cells

[0081] BTLA: B- and T lymphocyte attenuator; CTLA: Cytotoxic T lymphocyte antigen; HVEM: Herpes virus entry mediator; ICOS: Inducible T cell co-stimulator; ICOSL: Inducible T cell co-stimulator ligand; PD-1: Programmed death-1; PD-L: Programmed death ligand; PD-1H: Programmed death-1 homolog; VISTA: V domain Ig suppressor of T cell activation.

[0082] Table 2 Tumor necrosis factor receptor super family co-stimulatory molecules TNFR SF co- Function Cells expressing the Ligand Cells expressing the ligand stimulatory receptor molecules OX40 (CD134) Activation Activated and regulatory OX40L T cells, macrophages, T cells endothelial cells, vascular smooth muscle cells, dendritic cells, tumor cells CD27 (TNFR SF7) Activation T and B cells, NK cells CD70 NK, T and B cells GITR (CD357) Activation T cells GITRL T cells CD30 (TNFR SF8) Activation T and B cells CD30L T cells HVEM (CD270) Activation Monocytes, T and B LIGHT, Monocytes and APCs cells BTLA, CD160, LTα3, HSV1gD FAS (CD95) Activation NK and T cells FASL Dendritic cells, NK, T cells, neutrophils 19 162665565.1CD40 (TNFR SF5) Activation All B-cell lineages CD40L Activated CD4+ T cells, except plasma cells, some CD8+ T cells, γδ T macrophages, activated cells, basophils, platelets monocytes, follicular monocytes anddendritic cells, interdigitating dendritic cells, endothelial cells, fibroblasts RANK (CD265) Activation Osteoclast and dendritic RANKL Osteoblasts, T cells cells TACI (CD267) Inhibition B and plasma cells BAFF, Stromal cells, dendritic APRIL cells, and macrophages

[0083] APCs: Antigen-presenting cells; APRIL: A proliferation-inducing ligand; BAFF: B- cell activating factor; BTLA: B- and T lymphocyte attenuator; FASL: FAS ligand; GITR: Glucocorticoid-induced TNFR-related protein; HSV1gD: Herpes simplex virus-1 glycoprotein D; HVEM: Herpes virus entry mediator; LIGHT: TNFR14; LT ^3: Lymphotoxin-alpha 3; RANK: Receptor activator of nuclear factor kappa; TACI: Transmembrane activator and calcium- modulator and cyclophilin ligand interactor; TNFR SF: Tumor necrosis factor superfamily.

[0084] Cells of the Immune System

[0085] There are a large number of cellular interactions that comprise the immune system. These interactions occur through specific receptor-ligand pairs that signal in both directions so that each cell receives instructions based on the temporal and spatial distribution of those signals.

[0086] Murine models have been highly useful in discovering immunomodulatory pathways, but clinical utility of these pathways does not always translate from an inbred mouse strain to an outbred human population, since an outbred human population may have individuals that rely to varying extents on individual immunomodulatory pathways.

[0087] Cells of the immune system include lymphocytes, monocytes / macrophages, dendritic cells, the closely related Langerhans cells, natural killer (NK) cells, mast cells, basophils, and other members of the myeloid lineage of cells. In addition, a series of specialized epithelial and stromal cells provide the anatomic environment in which immunity occurs, often by 20 162665565.1secreting critical factors that regulate growth and / or gene activation in cells of the immune system, which also play direct roles in the induction and effector phases of the response.

[0088] The cells of the immune system are found in peripheral organized tissues, such as the spleen, lymph nodes, Peyer’s patches of the intestine and tonsils. Lymphocytes also are found in the central lymphoid organs, the thymus, and bone marrow where they undergo developmental steps that equip them to mediate the myriad responses of the mature immune system. A substantial portion of lymphocytes and macrophages comprise a recirculating pool of cells found in the blood and lymph, providing the means to deliver immunocompetent cells to sites where they are needed and to allow immunity that is generated locally to become generalized.

[0089] The term “lymphocyte” refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens through recombination of their genetic material (e.g. to create a T cell receptor and a B cell receptor). This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence of receptors specific for determinants (epitopes) on the antigen on the lymphocyte’s surface membrane. Each lymphocyte possesses a unique population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions.

[0090] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells).

[0091] B-Lymphocytes: B-lymphocytes are derived from hematopoietic cells of the bone marrow. A mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface. The activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help. In many physiological situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses. 21 162665565.1

[0092] Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors, because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes.

[0093] Cognate help allows B-cells to mount responses against antigens that cannot cross- link receptors and, at the same time, provides costimulatory signals that rescue B cells from inactivation when they are stimulated by weak cross-linkage events. Cognate help is dependent on the binding of antigen by the B-cell’s membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal / lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major histocompatibility complex (MHC) molecules. The resultant class II / peptide complexes are expressed on the cell surface and act as ligands for the antigen- specific receptors of a set of T-cells designated as CD4+T-cells. The CD4+T-cells bear receptors on their surface specific for the B-cell’s class II / peptide complex. B-cell activation depends not only on the binding of the T cell through its T cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B- cell activation. In addition, T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B-cell by binding to cytokine receptors on the B cell.

[0094] During cognate help for antibody production, the CD40 ligand is transiently expressed on activated CD4+T helper cells, and it binds to CD40 on the antigen-specific B cells, thereby transducing a second costimulatory signal. The latter signal is essential for B cell growth and differentiation and for the generation of memory B cells by preventing apoptosis of germinal center B cells that have encountered antigen. Hyperexpression of the CD40 ligand in both B and T cells is implicated in pathogenic autoantibody production in human SLE patients.

[0095] T-Lymphocytes: T-lymphocytes derived from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide range of immunologic functions. These include the capacity to help B cells develop into antibody- 22 162665565.1producing cells, the capacity to increase the microbicidal action of monocytes / macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on T cell expression of specific cell surface molecules and the secretion of cytokines.

[0096] Antigen Presenting Cells (APCs): Antigen-presenting cells fall into two categories: professional and non-professional. Those that express MHC class II molecules along with co- stimulatory molecules and pattern recognition receptors are often called professional antigen- presenting cells. The non-professional APCs express MHC class I molecules.

[0097] T cells must be activated before they can divide and perform their function. This is achieved by interacting with a professional APC which presents an antigen recognized by their T cell receptor. The APC involved in activating T cells is usually a dendritic cell. T cells cannot recognize (and therefore cannot respond to) "free" or soluble antigens. They can only recognize and respond to antigen that has been processed and presented by cells via carrier molecules like MHC molecules. Helper T cells can recognize exogenous antigen presented on MHC class II; cytotoxic T cells can recognize endogenous antigen presented on MHC class I. Most cells in the body can present antigen to CD8+cytotoxic T cells via MHC class I. Such cells express MHC class I and MHC class II molecules and can stimulate CD4+helper T cells as well as cytotoxic T cells. APCs can also present foreign and self lipids to T cells and NK cells by using the CD1 family of proteins, which are structurally similar to the MHC class I family.

[0098] Professional APCs: Professional APCs specialize in presenting antigens to T cells. They are very efficient at internalizing antigens, either by phagocytosis (e.g. macrophages), or by receptor-mediated endocytosis (B cells), processing the antigen into peptide fragments and then displaying those peptides (bound to a class II MHC molecule) on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen-presenting cell. An additional co-stimulatory signal is then produced by the antigen- presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules and MHC class II are defining features of professional APCs. All professional APCs also express MHC class I molecules as well.

[0099] The main types of professional antigen-presenting cells are dendritic cells, macrophages and B cells. 23 162665565.1[000100] Dendritic cells (DCs): Dendritic cells have the broadest range of antigen presentation and are necessary for activation of naive T cells. DCs present antigen to both helper and cytotoxic T cells. They can also perform cross-presentation, a process by which they present exogenous antigen on MHC class I molecules to cytotoxic T cells. Cross-presentation allows for the activation of these T cells. Dendritic cells also play a role in peripheral tolerance, which contributes to prevention of auto-immune disease. [000101] Prior to encountering foreign antigen, dendritic cells express very low levels of MHC class II and co-stimulatory molecules on their cell surface. These immature dendritic cells are ineffective at presenting antigen to T helper cells. Once a dendritic cell's pattern-recognition receptors recognize a pathogen-associated molecular pattern, antigen is phagocytosed and the dendritic cell becomes activated, upregulating the expression of MHC class II molecules. It also upregulates several co-stimulatory molecules required for T cell activation, including CD40 and B7. The latter can interact with CD28 on the surface of a CD4+T cell. The dendritic cell is then a fully mature professional APC. It moves from the tissue to lymph nodes, where it encounters and activates T cells. [000102] Classical DCs (cDCs) and pre-plasmacytoid DCs (pDCs) are derived from the common lymphoid progenitor (CLP) and common myeloid (CMP) pathways that originate in bone marrow and terminate as blood borne monocytes that develop into mature DCs. [000103] cDCs are highly migratory and are found at sites of likely microbial encounters such as the gut, skin and lungs. The immature cDCs capture and process antigen then transit from these tissues to lymphoid organs such as spleen and lymph nodes where they mature, up-regulate co-stimulatory molecules and present the antigen in the context of a MHC-II-peptide complex to lymphocytes. [000104] pDCs are also found in peripheral organs, primarily those of the lymphatic system in a steady state. The fundamental function of pDCs is to respond to viral infection, by the secretion of type I interferon alpha / beta; however they can also be involved in both the activation and control of T cell responses. [000105] The conventional DC (cDC), sometimes also referred to as classical DC or myeloid DC, travels through the blood to differentiate within lymphoid and non-lymphoid tissues. This DC subset features irregularly-shaped cells with many cell membrane processes. In 24 162665565.1particular, they are known to be specialized for absorbing antigens for presentation to naïve T cells in adaptive immunity. cDCs are further categorized into two major subpopulations, cDC1 and cDC2. [000106] Prominent cDC1 markers include CD8A, CLEC9A, ITGAE, ITGAX, THBD (CD141), and XCR1. [000107] cDC2s are a dominant subset of DCs residing in the blood, tissues, and lymphoid organs. They express a wide repertoire of pattern recognition receptors, including Toll-like receptors and lectins, that drive innate immune responses. They have been shown to induce Th2 and Th17 helper T cell responses and a role in inducing of CD4+T cell responses. cDC2s appear to have many regulatory roles, such as inducing regulatory T cells in the intestine and maintaining tolerance in the liver. Human cDC2s are understood to be heterogeneous and have been recently suggested to include more distinct subtypes. Common cDC2 markers include CD1C, CD207, ITGAM, NOTCH2, and SIRPA. [000108] Plasmacytoid dendritic cells (pDCs) mature directly from CDPs within the bone marrow before migrating to the blood and peripheral lymphoid tissues. These round, plasma cell- shaped cells are less abundant than their conventional counterparts. pDCs are notable for being able to rapidly produce type I and type III interferons and cytokines, making this an important subset for responding to acute or chronic viral infection. pDCs express MHC II, potentially being able to act as antigen-presenting cells. Subsets also exist among the pDCs. One population, defined by high expression of CD2, has been found to exclusively induce CD4+T cell proliferation. Upon stimulation, pDCs have been reported to also activate CD8+ T cells. Markers for pDCs include CLEC4C, LILRB4, NRP1, CCR7, B220 (mouse), and SiglecH (mouse). [000109] Monocyte-derived dendritic cells (mo-DC) —This subset, featuring a dendritic morphology, also shares gene signatures of the in vitro monocyte-derived DCs (mo-DCs). mo-DC is produced in response to inflammation and that this subset promotes CD4+T cells to produce a Th17 immune response. Some markers reported for mo-DCs include CD14, MRC1 (CD206), CD209, SIRPA, ITGAM (CD11b), and CD1A. [000110] Langerhans cells (LC) — LC are immune cells residing in areas of stratified squamous epithelium, such as the skin. They are macrophages in origin but have also been considered to be dendritic cell-like due to phenotype. Like dendritic cells, LC can extend 25 162665565.1dendrites, absorb foreign antigens, travel to the lymph node, and then present the antigen to T cells. Langherans markers include CD1A, CD207 (Langerin), and ID2. [000111] AXL+Siglec6+ dendritic cells (AS DC) — A population of pre-DC in the blood appears to form a distinct, functional subset that is distinct from pDCs and cDCs. Identified by the expression of two key markers, AXL and Siglec6 (CD327), this subset is reported to strongly stimulate T cells in lymphoid tissue. It is noted that AS DCs may also be a mature cDC2s precursor and may potentially transition toward this subset. [000112] Accordingly, in some embodiments, an artificial antigen presenting cell (aAPC) engineered to activate T cells, comprises dendritic cell markers. In certain aspects, dendritic cell markers comprise markers of plasmacytoid dendritic cells (pDC), conventional dendritic cells 1 (cDC1), conventional dendritic cells 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof. [000113] In certain aspects, the aAPC comprises one or more dendritic cell markers. In certain embodiments, the dendritic cell markers comprise BDCA-1, CD8, CD8alpha, CD11b, CD11c, CD103, CD205, MHC Class II, BDCA-2, BDCA-4, CD11clowCD45RA, CD123 ILT-7, MHC Class IIlowTLR7, TLR9 or combinations thereof. [000114] Table 3: Human Dendritic Markers 26 162665565.1Gene Synonyms Marker Type Protein Type Localization Size (kDa)27 162665565.1[000115] Macrophages: Macrophages can be stimulated by B cells secretion of interferon. After this activation, macrophages are able to express MHC class II and co-stimulatory molecules, including the B7 complex and can present phagocytosed peptide fragments to helper B cells. Activation can assist pathogen-infected macrophages in clearing the infection. Deriving from monocytes, a type of white blood cell, they will circulate in the blood and enter affected sites and differentiate from monocytes to macrophages. At the affected site, the macrophage surrounds the site of infection or tissue damage with its membrane in a mechanism called phagocytosis. Accordingly, in some embodiments, an artificial antigen presenting cell (aAPC) engineered to activate T cells, comprises a macrophage cell phenotype. [000116] In some aspects, an artificial antigen presenting cell (aAPC) engineered to activate T cells, comprises a macrophage cell phenotype. [000117] According to the activation state and functions of macrophages, they can be divided into M1-type (classically activated macrophage) and M2-type (alternatively activated macrophage). [000118] Table 4 General Markers for M1 and M2 macrophages Gene Synonyms Species Marker Type Protein Type Localization Size (kDa) 97.7 41.9 40.137.4 108 14.2 52.7166131.1 57.6182.689.834.728 162665565.1CD163^ Hu, Mo M2, Monocyte, Tissue Receptor Cell Membrane 125.5(perivascular, Kupffer)CD200R1^ CD200R, OXR Hu, Mo Tissue (alveolar) Receptor Cell Membrane, 36.6Secreted33 37.7 35.4 27.616.3 40.442.6127.2 110.2 31 11.9 25.610.653.1 30.620.5 30.7ted23.7 sm, 26.295.7 11 40.5 45.825.637.7 60.2 11.4 29.119.3 17.5 51.8127.849.8, , 27.3 29 162665565.1PTPRC^ CD45, LCA Hu, Mo Tissue (adipose-associated, Receptor Cell Membrane 147.5perivascular, meningeal)STAT6^ Hu, Mo M2 Transcription Nucleus, Cytoplasm 94.1factor41.6 44.52465.4 84.8 31 120.9macrophages exhibit a pro-inflammatory phenotype and produce cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6, critical for host defense against pathogens. [000120] M1 macrophages mainly function in Th1 cell recruitment, pathogen resistance, and tumor control via innate and adaptive immune responses. The polarization into M1 macrophages can be classically activated by pathogens, LPS, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α), and the T helper 1 (Th1) cell cytokine interferon gamma (IFN-γ). To drive macrophages to M1 polarization, many pathways are involved, including the IRF / STAT, LPS / TLR4, and NF-κB / PI-3 kinase pathways. [000121] Characteristically, M1 macrophages show high antigen presentation activity and high production of pro-inflammatory cytokines, such as interleukin 1 (IL-1), IL-6, TNF-α, and of nitric oxide (NO) and reactive oxygen species (ROS). Furthermore, they display an upregulation of IL-12 and IL-23 and a downregulation of IL-10. Stimulation of M1 macrophages results in high secretion levels of IL-1β, TNF-α, IL-12, IL-18, and IL-23. Moreover, the M1 macrophage phenotype has been shown to express high levels of major histocompatibility complex class II (MHC II), CD68, CD80, and CD86, as well as of the Th1 cell-attracting chemokines, including CXCL9 and CXCL12. [000122] Table 5. Human M1 macrophage markers. 30 162665565.1Species Macrophage type Marker Marker type31 162665565.1Human M1 Mer (MerTK) Surface n n[000123] Alternatively activated macrophages (M2): These macrophages are induced by interleukin-4 (IL-4), IL-13, or IL-10, and are typically associated with tissue repair and remodeling. M2 macrophages produce anti-inflammatory cytokines such as IL-10 and 32 162665565.1Species Macrophage type Marker Marker type H M2 IDO S d cription cription -2,3- r of ng 4.162665565.1[000124] transforming growth factor-beta (TGF-β) and extracellular matrix proteins that facilitate tissue repair. [000125] M2 macrophages can be alternatively activated by parasitic or fungal infection, immune complexes, apoptotic cells, macrophage colony-stimulating factor (M-CSF), IL-13, TGF- β, and T helper 2 (Th2) cytokine IL-4, as well as by IL-33 and IL-25 via Th2 cells. Underlying signaling that drives macrophages into the M2 state involves STAT6, IRF4, PPARδ, and PPARγ. [000126] In contrast to the classically activated subtype, the alternatively activated subtype shows the opposite expression profile: downregulation of IL-12 and IL-23 and upregulation of IL- 10 and IL-1RA. In addition, M2 macrophages show low production of the inflammatory cytokines IL-1, IL-6, and TNF-α. M2 macrophages function in pathogen clearance, anti- inflammatory response, and metabolism, as well as in wound healing, tissue remodeling, immunoregulation, tumor progression, and malignancies. [000127] The M2 phenotype can be characterized by the expression of CD206, CD163, CD209, FIZZ1 and Ym1 / 2. In general, this subtype shows high expression of receptors required for the phagocytosis and scavenging of mannose and galactose, as well as high production of ornithine and polyamines via the arginase pathway. Chemokines expressed by this macrophage type are CCL1, CCL17, CCL18, CCL22, and CCL24. [000128] Subtypes of M2 macrophages: Dependent on the respective stimulation of M2 macrophages, four M2 subtypes have been established: M2a, M2b, M2c, and M2d. These subtypes differ from each other based on their cell surface markers, secreted cytokines, and biological functions. However, all M2 macrophage subtypes have IL-10 expression in common. [000129] M2a macrophages: Activation by IL-4 or IL-13. IL-4 in turn leads to the expression of the mannose receptor (CD206). Further upregulation of IL-10, TGF-β, CCL17, CCL18, and CCL22 have been shown to promote cell growth, tissue repair, and endocytosis. [000130] M2b macrophages: Activation by immune complexes, Toll-like receptor (TLR) ligands and IL-1b. Upon activation, this subtype releases pro- and anti-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-10. M2b macrophages function in the regulation of the immune response and inflammation. [000131] M2c macrophages: Activation by glucocorticoids, IL-10, and TGF-β (and inactivated macrophages). This subtype is characterized by high expression of anti-inflammatory 34 162665565.1IL-10, pro-fibrotic TGF-β, CCL16, CCL18, as well as the Mer receptor tyrosine kinase (MerTK), which promotes phagocytosis of apoptotic cells. [000132] M2d macrophages: Activation by TLR antagonists, IL-6, and adenosines. Adenosines lead to the expression of IL-10 and vascular endothelial growth factor (VEGF), which in turn triggers angiogenesis and tumor progression. [000133] In certain aspects, the artificial antigen presenting cell (aAPC) comprises one or more macrophage markers. In certain embodiments, the macrophage markers comprise M1 macrophage markers, M2 macrophage markers or combinations thereof. In certain embodiments, the M1 macrophage markers comprise CD86, CD80, CD68, MHC class II, IL-1R, TLR2, TLR4, iNOS, SOCS or combinations thereof. In certain embodiments, the M2 macrophage markers comprise CD163, MHC class II, SR, MMR / CD206, CD200R, TGM2, DecoyR, IL-1R II, CD86, CD163, TLR1, TLR8, VEGF or combinations thereof. [000134] Table 6: Human M2 subtype macrophage markers. M2a M2b M2c M2d CD163 CD86 CD163 VEGF[000135] B cells: B cells can internalize antigen that binds to their B cell receptor and present it to helper T cells. Unlike T cells, B cells can recognize soluble antigen for which their B cell receptor is specific. They can then process the antigen and present peptides using MHC class II molecules. When a T helper cell with a TCR specific for that peptide binds, the B cell marker CD40 binds to CD40L on the T cell surface. When activated by a T cell, a B cell can undergo antibody isotype switching, affinity maturation, as well as formation of memory cells. [000136] Non-professional APCs: Non-professional antigen presenting cells include all nucleated cell types in the body. They use an MHC class I molecule coupled to β2 microglobulin 35 162665565.1to display endogenous peptides on the cell membrane. These peptides originate within the cell itself, in contrast to the exogenous antigen displayed by professional APCs using MHC class II molecules. Cytotoxic T cells are able to interact with endogenous antigen presented using an MHC class I molecule. Non-professional APCs do not typically express MHC class II molecules. However, it has been observed that antigen presentation to CD4+cells via MHC class II is not restricted to the classically professional APCs. Other leukocytes, including granulocytes such as mast cells and neutrophils, can be induced to do so, as can endothelial and epithelial cells under certain circumstances. [000137] Antigen recognition: Immunoglobulin, the B cell’s receptor, binds to individual epitopes on soluble molecules or on particulate surfaces. B-cell receptors see epitopes expressed on the surface of native molecules. While antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids, T cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen- presenting cells (APCs). There are three main types of APCs in peripheral lymphoid organs that can activate T cells: dendritic cells, macrophages and B cells. The most potent of these are the dendritic cells, whose only function is to present foreign antigens to T cells. [000138] Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and carry them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an APC that can activate T cells. APCs display three types of protein molecules on their surface that have a role in activating a T cell to become an effector cell: (1) MHC proteins, which present foreign antigen to the T cell receptor; (2) costimulatory proteins which bind to complementary receptors on the T cell surface; and (3) cell- cell adhesion molecules, which enable a T cell to bind to the APC for long enough to become activated. T-cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T cells express T cell receptors (TCR) consisting of α and β- chains. A small group of T cells express receptors made of γ and δ chains. Among the α / β T cells are two sub-lineages: those that express the coreceptor molecule CD4 (CD4+T cells); and those that 36 162665565.1express CD8 (CD8+T cells). These cells differ in how they recognize antigen and in their effector and regulatory functions. [000139] CD4+T cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T cells are activated, and the wide array of cytokines they secrete when activated. T cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms. In addition, T cells, particularly CD8+T cells, can develop into cytotoxic T- lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs. [000140] T cell receptors (TCRs) recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class I MHC protein. CD4+T cells recognize only peptide / class II complexes while CD8+T cells recognize peptide / class I complexes. The TCR’s ligand (i.e., the peptide / MHC protein complex) is created within APCs. In general, class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide-loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4+T cells with TCRs capable of recognizing the expressed cell surface complex. Thus, CD4+ T cells are specialized to react with antigens derived from extracellular sources. [000141] In contrast, class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally composed of nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+T cells expressing appropriate receptors. This gives the T cell system, particularly CD8+ T cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., viral antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted. 37 162665565.1[000142] T cells can also be classified based on their function. Among the sub-types and subpopulations of T cells and / or of CD4+and / or of CD8+T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCMXcentral memory T (TCM effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. [000143] Helper T cells are T cells that stimulate B cells to make antibody responses to proteins and other T cell-dependent antigens. T cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so inefficiently. B cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes, and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting peptide / class II MHC complex is then exported to the B-cell surface membrane. T cells with receptors specific for the peptide / class II molecular complex recognize this complex on the B-cell surface. [000144] B-cell activation depends both on the binding of the T cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B cell. T cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T cell and CD80 or CD86. CD80 / CD86 is generally expressed by activated, but not resting, B cells so that the helper interaction involving an activated B cell and a T cell can lead to efficient antibody production. In many cases, however, the initial induction of CD40L on T cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80 / 86, such as dendritic cells. Such activated helper T cells can then efficiently interact with and help B cells. Cross-linkage of membrane Ig on the B cell, even if inefficient, may synergize with the CD40L / CD40 interaction to yield vigorous B-cell activation. The subsequent events in the B-cell 38 162665565.1response, including proliferation, Ig secretion, and class switching of the Ig class being expressed, either depend or are enhanced by the actions of T cell-derived cytokines. [000145] CD4+T cells tend to differentiate into cells that principally secrete the cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that mainly produce IL-2, IFN-γ, and lymphotoxin (TH1 cells). The TH2 cells are very effective in helping B-cells develop into antibody-producing cells, whereas the TH1 cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments. Although CD4+T cells with the phenotype of TH2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, TH1cells also have the capacity to be helpers. [000146] T cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFN-γ) produced by helper T cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production. TH1 cells are effective in enhancing the microbicidal action, because they produce IFN-γ. In contrast, two of the major cytokines produced by THH2 cells, IL-4 and IL-10, block these activities. [000147] Regulatory T cells (Tregs): Tregs are important in the maintenance of immune cell homeostasis as evidenced by undesirable consequences of genetic or physical ablation of the Treg population. Treg cells generally maintain order in the immune system by enforcing a dominant negative regulation on other immune cells. Broadly classified into natural or adaptive (induced) Tregs; natural Tregs are CD4+CD25+T-cells which develop and emigrate from the thymus to play a role in immune homeostasis. Adaptive Tregs are non-regulatory CD4+T-cells which acquire CD25 (IL-2R alpha) expression outside of the thymus, and may be induced by inflammation and disease processes, such as autoimmunity and cancer. [000148] There is increasing evidence that Tregs acquire their function through a myriad of mechanisms that may include the secretion of immunosuppressive soluble factors such as IL-9, IL-10 and TGF beta, cell contact mediated regulation via the high affinity TCR and other costimulatory molecules such as CTLA-4, GITR, and cytolytic activity. Under the influence of TGF beta, adaptive Treg cells mature in peripheral sites, including mucosa-associated lymphoid 39 162665565.1tissue (MALT), from CD4+Treg precursors, where they acquire the expression of markers typical of Tregs, including CD25, CTLA4 and GITR / AITR. Upon up-regulation of the transcription factor Foxp3, Treg cells begin their suppressive effect. This includes the secretion of cytokines including IL-10 and TGF beta which may induce cell-cycle arrest or apoptosis in effector T cells and blocking co-stimulation and maturation of dendritic cells. [000149] Cytotoxic T Lymphocytes: CD8+T cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target cells. The mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that cell. Perforin-mediated lysis is enhanced by granzymes, a series of enzymes produced by activated CTLs. Many active CTLs also express large amounts of FAS ligand on their surface. The interaction of fas ligand on the surface of CTL with FAS on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these cells. CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells. [000150] Lymphocyte Activation: The term “activation” or “lymphocyte activation” refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells. T-cell activation is dependent on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adapter proteins that link the TCR to the Ras pathway, phospholipase Cγ1, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. Full responsiveness of a T cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and / or CD86 on the APC. T-memory Cells 40 162665565.1[000151] Following the recognition and eradication of pathogens through adaptive immune responses, the vast majority (90–95%) of T cells undergo apoptosis with the remaining cells forming a pool of memory T cells, designated central memory T cells (TCM), effector memory T cells (TEM), and resident memory T cells (TRM) (Clark, R.A., “Resident memory T cells in human health and disease”, Sci. Transl. Med., 7, 269rv1, (2015)). CD45RA is expressed on naïve T cells, as well as the effector cells in both CD4 and CD8. After antigen experience, central and effector memory T cells gain expression of CD45RO and lose expression of CD45RA. Thus either CD45RA or CD45RO is used to generally differentiate the naïve from memory populations. CCR7 and CD62L are two other markers that can be used to distinguish central and effector memory T cells. Naïve and central memory cells express CCR7 and CD62L in order to migrate to secondary lymphoid organs. Thus, naïve T cells are CD45RA+CD45RO–CCR7+CD62L+, central memory T cells are CD45RA–CD45RO+CCR7+CD62L+, and effector memory T cells are CD45RA–CD45RO+CCR7–CD62L–. [000152] Compared to standard T cells, these memory T cells are long-lived with distinct phenotypes such as expression of specific surface markers, rapid production of different cytokine profiles, capability of direct effector cell function, and unique homing distribution patterns. Memory T cells exhibit quick reactions upon re-exposure to their respective antigens in order to eliminate the reinfection of the offender and thereby restore balance of the immune system rapidly. Increasing evidence substantiates that autoimmune memory T cells hinder most attempts to treat or cure autoimmune diseases (Clark, R.A., “Resident memory T cells in human health and disease”, Sci. Transl. Med., Vol.7, 269rv1, (2015)). [000153] Accordingly, In another aspect, the present disclosure provides an artificial antigen presenting cell (aAPC) engineered to activate T cells, the aAPC comprising an exogenous antigen presenting poly peptide and an exogenous antigen-presenting polypeptide on the cell surface, wherein the exogenous antigen-presenting polypeptide is an MHC class I single-chain fusion protein or an MHC class II single-chain fusion protein. [000154] In certain embodiments, the MHC class I single chain fusion protein comprises an α chain and a β2m chain. In certain embodiments, the MHC class I single chain fusion protein further comprises a membrane anchor. In certain embodiments, the foreign antigen polypeptide is connected to the MHC I single chain fusion protein via a linker. In certain embodiments, the 41 162665565.1linker is a cleavable linker. In certain embodiments, the MHC class II single chain fusion protein comprises an α chain and a β chain. In certain embodiments, the MHC class II single chain fusion protein further comprises a membrane anchor. In certain embodiments, the foreign antigen polypeptide is connected to the MHC II single chain fusion protein via a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the anchor comprises glycophorin A (GPA) protein or comprises the transmembrane domain of small integral membrane protein 1 (SMIM1). In certain embodiments, the foreign antigen polypeptide is covalently linked to the foreign antigen-presenting polypeptide. In certain embodiments, the foreign antigen polypeptide is non-covalently bound to the foreign antigen-presenting polypeptide. [000155] In some embodiments, the aAPC comprises a positive co-stimulatory molecule. In certain embodiments, the positive co-stimulatory molecule is a TNF receptor superfamily member. In some embodiments, the TNF receptor superfamily member is selected from the group consisting of: CD120a, CD120b, CD18, OX40, CD40, Fas receptor, M68, CD27, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, RANK, OCIF, TWEAK receptor, TACI, BAFF receptor, ATAR, CD271, CD269, AITR, TROY, CD358, TRAMP, and XEDAR. In some embodiments, the activator for a positive co-stimulatory molecule is a TNF superfamily member. In some embodiments, the TNF superfamily member is selected from the group consisting of: TNFα, TNF-C, OX40L, CD40L, FasL, LIGHT, TL1A, CD27L, Siva, CD153, 4-1BB ligand, TRAIL, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF, BDNF, NT-3, NT-4, GITR ligand, and EDA-2. In some embodiments, the positive co-stimulatory molecule is a CD28-superfamily co-stimulatory molecule. In some embodiments, the CD28-superfamily co-stimulatory molecule is ICOS or CD28. In some embodiments, the activator for a positive co-stimulatory molecule is ICOSL, CD80, or CD86. [000156] In certain embodiments, the aAPC further comprises at least one exogenous co- stimulatory polypeptide on the cell surface. In certain embodiments, the at least one exogenous co-stimulatory polypeptide is 4-1BBL, LIGHT, anti-CD28, CD80, CD86, CD70, OX40L, GITRL, TIM4, SLAM, CD48, CD58, CD83, CD155, CD112, IL-15Rα fused to IL-15, IL-21, ICAM-1, ligand of LFA-1, anti-CD3, and combinations thereof. In certain embodiments, the 42 162665565.1aAPC comprises at least 2, at least 3, at least 4, or at least 5 exogenous co-stimulatory polypeptides on the cell surface. [000157] In certain embodiments, the aAPC further comprises an exogenous cytokine polypeptide on the cell surface. In certain embodiments, the exogenous cytokine polypeptide is IL-2, IL-15, 15Rα fused to IL-15, IL-7, IL-12, IL-18, IL-21, IL-4, IL-6, IL-23, IL-27, IL-17, IL- 10, TGF-β, IFNγ, IL-1β, GM-CSF, and IL-25. [000158] In certain embodiments, aAPCs activate T cells comprising CD8+T cells, CD4+T cells. In certain embodiments, the aAPCs stimulate T cell cytotoxic activity, stimulate cytokine secretion by T cells, and / or any combination thereof. [000159] In certain embodiments, the artificial antigen-presenting cells (aAPCs) are engineered to suppress T cell activity e.g. in the treatment of autoimmune diseases. In certain embodiments, the aAPC comprises an antigen-presenting polypeptide and at least one co- inhibitory polypeptide on the cell surface. [000160] In certain embodiments, the aAPC regulate regulatory T cell (Treg) responses. [000161] In certain embodiments, the aAPC comprises an inhibitory polypeptide. In some embodiments, the inhibitory polypeptide is IL-35, IL-10, VSIG-3, PD-L1 or a LAG3 agonist. [000162] In certain embodiments, the aAPC further comprises encapsulating a peptide, a nucleic acid, a cytokine, an adjuvant, a therapeutic agent, a toxin, or combinations thereof. [000163] Methods of Treatment [000164] In certain embodiments, a method of treating a subject requiring immunotherapy such as for example, cancer, autoimmunity, and the like comprises administering to the subject a therapeutically effective amount of aAPCs embodied herein. In certain embodiments, an aAPC with a positive co-stimulatory signal is administered to a patient diagnosed as having cancer or is infected with an infectious agent. In certain embodiments, an aAPC with a negative signal is administered to a subject with an autoimmune disease. [000165] The composition of the present disclosure may be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents. 43 162665565.1[000166] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents. [000167] The carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the aAPCs. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers. [000168] In an embodiment, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration. [000169] Compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. [000170] The precise amount of aAPCs to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject. [000171] Compositions comprising the aAPCs of the present disclosure may also be administered multiple times at these dosages. The aAPCs can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., 1988, New England Journal of Medicine, 319: 1676). The optimal dosage and treatment regimen for a particular 44 162665565.1subject can be readily determined by one skilled in the art by monitoring the patient for signs of disease and adjusting the treatment accordingly. [000172] Combination Therapies [000173] The disclosure also contemplates the combination of the composition of the present disclosure with other drugs and / or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. For example, in the case of cancer chemotherapeutic agents may be administered as part of the combination therapy. [000174] In the case of autoimmune diseases, a combination therapy may include aAPCs and one or more anti-inflammatory agents and / or therapeutic agents. The one or more anti- inflammatory agents and / or therapeutic agents may be administered simultaneously or sequentially. The anti-inflammatory agents comprise one or more antibodies which specifically bind to pro-inflammatory cytokines, e.g. pro-inflammatory cytokines such as IL-1, IL-4, IL-5, IL- 6, IL-8, IL-10, GM-CSF, TNFα IFN-γ and MIP-1a. In certain embodiments, the antibodies are anti-TNFα, anti-IL-6 or combinations thereof. In certain embodiments, one or more agents, other than antibodies can be administered which decrease pro-inflammatory cytokines, e.g. non- steroidal anti-inflammatory drugs (NSAIDs). Any combination of antibodies and one or more agents can be administered which decrease pro-inflammatory cytokines. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti- inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. [000175] In certain embodiments, the aAPCs contemplated herein are administered in conjunction with a cytokine. [000176] In certain embodiments, the aAPCs are administered in conjunction with a cancer therapy. As used herein, the term “cancer therapy” refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, 45 162665565.1chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTINTM), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVATM)), platelet derived growth factor inhibitors (e.g., GLEEVECTM(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s), TRAIL / Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also contemplated for use with the methods described herein. [000177] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVATM, Genentech / OSI Pharm.), Bortezomib (VELCADETM, Millennium Pharm.), Fulvestrant (FASLODEXTM, Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARATM, Novartis), Imatinib mesylate (GLEEVECTM, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (EloxatinTM, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNETM, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSATM, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXANTMcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozcicsin, carzcicsin and bizcicsin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, 46 162665565.1lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1 and calicheamicin omega 1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINTMdoxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, strcptonigrin, strcptozocin, tubcrcidin, ubenimcx, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKTMpolysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosinc; arabinoside (“Ara-C”); cyclophosphamidc; thiotcpa; taxoids, e.g., TAXOLTMpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANETMCremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERETMdoxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZARTM47 162665565.1gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINETMvinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. [000178] Also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEXTM(tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTONTM(toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASETM(megestrol acetate), AROMASINTM(exemestane), formestanie, fadrozole, RIVISORTM(vorozole), FEMARATM(letrozole), and ARIMIDEXTM(anastrozole); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYMETM(ribozyme)) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTINTMvaccine, LEUVECTINTMvaccine, and VAXIDTMvaccine; PROLEUKINTMrIL-2; LURTOTECANTMtopoisomerase 1 inhibitor; ABARELIXTMrmRH; (x) anti-angiogenic agents such as bevacizumab (AVASTINTM, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above. [000179] In various embodiments, the cancer therapeutic is an immunotherapy selected from the group comprising oncolytic virus, bacteria, oncolytic bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), a microbiome modulator, and / or a toll-like receptor (TLR) agonist. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonist. In various embodiments, the TLR agonist is 48 162665565.1derived from virus, bacteria and / or made synthetically. In various embodiments, the immunotherapy is a is a stimulator of interferon genes (STING) pathway modulator. [000180] It will be appreciated by those skilled in the art of cancer immunotherapy that other complementary immune therapies may be added to the regimens described above to further enhance their efficacy including but not limited to GM-CSF to increase the number of myeloid derived innate immune system cells, low dose cyclophosphamide or PI3K inhibitors (e.g., PI3K delta inhibitors) to eliminate T regulatory cells that inhibit innate and adaptive immunity and 5FU (e.g., capecitabine), PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells. For example, PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC- 0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some aspects, the PI3K inhibitor is a PI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503. Additional PI3K inhibitors are disclosed in U.S. Patent Application Nos. US20150291595, US20110190319, and International Patent Application Nos. WO2012146667, WO2014164942, WO2012062748, and WO2015082376. The immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INFα. [000181] In certain embodiments, the aAPCs are administered with one or more immune checkpoint modulators. Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses. Examples of checkpoint inhibitor include, without limitation an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β. [000182] The term “checkpoint inhibitor” means a group of molecules on the cell surface of CD4+and / or CD8+T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012 / 177624). “Anti-immune checkpoint inhibitor therapy” refers to the use of 49 162665565.1agents that inhibit immune checkpoint inhibitors. Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. Exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and / or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and / or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fe portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, either alone or used in combination. [000183] In some embodiments, such therapy involves blockade of programmed cell death 1 (PD-1). In some embodiments, such therapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1). In some embodiments, such therapy involves administration of an antibody agent that specifically interacts with PD-1 or with PD-L1. In some embodiments, such therapy involves administration of one or more of nivolumab (BMS-936558, 50 162665565.1MDX-1106, ONO-4538, a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, a humanized monoclonal IgG4 anti-PD-1 antibody), BMS-936559 (a fully human IgG4 PD-L1 antibody), MPDL3280A (a humanized engineered IgG1 monoclonal PD-L1 antibody) and / or MEDI4736 (a humanized engineered IgG1 monoclonal PD-L1 antibody). [000184] The present systems are further disclosed in M. Omotoso, Alginate-based artificial antigen presenting cells expand functional CD8+T cells with memory characteristics for adoptive cell therapy, Biomaterials 2024 Aug 24:313:122773 doi: 10.1016 / j.biomaterials.2024.122773. [000185] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the disclosure. [000186] This disclosure is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are hereby incorporated by reference. EXAMPLES [000187] Example 1: Fabrication of Alginate Based aAPCs [000188] Step 1: Preparation of alginate nanoparticles [000189] Materials ^ 5 mL sterile water ^ 100 mg of sodium alginate ^ Sonicator (amplitude 60) ^ Homogenizer ^ 100 mL cyclohexane ^ 1M calcium chloride (CaCL2 in water) ^ 4500mg Span 80 ^ 1500mg Tween 20 [000190] Alginate preparation (water / oil phase / surfactant) Water (Alginate) (for 5mL total): 1. Place 5mL water on 90C hot stir plate to heat up 2. Weigh out 100mg alginate and add (slowly) to the hot 5mL DH20 3. let heat on heated stir plate for ~10 min or until alginate dissolves Oil Phase / Surfactant (nanosized): 51 162665565.11. Weigh out 4500mg of Span80 slowly with a plastic dropper 2. Tare and weigh out 1500mg of Tween80 slowly in the same small beaker 3. Add 100 mL cyclohexane and stir bar 4. Put oil and surfactant solution on stir plate at 500rpm 5. Put oil and surfactant solution on stir plate at 500rpm until completely combined. 6. Remove stir bar. Oil Phase / Surfactant (microsized): 1. Weigh out 1500mg of Span80 slowly with a plastic dropper 2. Tare and weigh out 500mg of Tween80 slowly in the same small beaker 3. Add 100 mL cyclohexane and stir bar 4. Put oil and surfactant solution on stir plate at 500rpm until completely combined. 5. Remove stir bar. [000191] Emulsify and crosslink 1. Place oil and surfactant solution on ice and sonicate for 1 min at 60 amp for nanosized particles. (5000 rpm for micros) 2. Add alginate solution and allow to sonicate for an additional 1 min at 60 amp. (5000 rpm for micros) 3. Add ~3 mLs (3 droppers full) of 1 M CaCl2 solution and continue to sonicate for 2 full minutes. 4. Pour out the particle solution evenly into four 50 mL tubes and top off with water, spin at 100xg to remove any large bits. 5. Slowly pour out the particle mixture into new centrifuge tubes until the line between the particles and the water shows. It is key that you don’t pour in the “line” of particles between the solution and the water. 6. Spin down at 21000xg. 7. Discard the supernatant. 8. Resuspend in ethanol by sonicating at 30 amp. 9. Spin down at 21000xg. 10. Resuspend in DI water and sonicate. Spin down at 21000xg. Repeat once more. 11. Resuspend in 5mL of DI H20. [000192] Sizing 1. place 990uL of DH20 in a zetasizer cuvette 2. add 10uL of particle solution into cuvette and mix 3. measure size 3x on the zetasizer [000193] Finding the concentration 52 162665565.11. Weigh a 500mL eppendorf tube and record the empty weight 2. Mix and pipette 150uL of particle solution into the tube 3. Leave tube open and place carefully in 50mL tube with no lid 4. Wrap a kimwipe across the top of the tube and secure with a rubber band 5. Place in -80C and freeze for 30 min (should be fully frozen) 6. Place in lyophilizer overnight 7. The next day, remove from lyophilizer and record weights of tubes. 8. Back-calculate particle concentration and dilute to 5mg / mL 9. Make 5mg aliquots at 5mg / mL and freeze at -20C [000194] Step 2: Surface protein conjugation for aAPC fabrication [000195] Materials ^ Sterile water ^ 2-(N-morpholino)ethanesulfonic acid (MES) ^ MES Buffer ^ 787 mg MES ^ Fill tube up to 40 mL with milliQ water ^ Add 2.5 mL of 1M NaOH. Test the pH using a pH strip and add NaOH in 500 uL increments until the pH is 6.0 ^ Sodium Hydroxide (NaOH) ^ N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) ^ N-hydroxysuccinimide (NHS) ^ Peptide loaded MHC-Ig ^ Anti-CD28 ^ 1X PBS [000196] Protein conjugation 1. Take a 5 mg aliquot, thaw, and spin down at 21000xg. 2. Remove the DI water. 3. Resuspend with 900 uL of MES buffer. 4. Prepare EDC / NHS a. 100 uL of EDC / NHS in MES buffer per 2.5 mgs or particles b. Weigh out the appropriate amounts of EDC and NHS: i. 40 mg EDC and 48 mg NHS per 1 mL of MES buffer 5. Quickly add 100 uL to particles. 6. Put the particles on the inverter at room temperature for 30 minutes. 7. Make the required protein solution: a. 5 ug of each protein per mg of particles in 1X PBS 8. Take tubes off inverter and spin at 21000xg for nanos to remove the supernatant. 9. Resuspend in 1 mL PBS: 53 162665565.1a. Vortex to resuspend microparticles b. Sonicate with the microtip at 30% amplitude for 5 seconds to resuspend nanoparticles 10. Wash 2 more times with 1X PBS. 11. After the final wash resuspend in 1 mL of 1X PBS. 12. Add protein solution and put on the inverter in the cold room. 13. Allow the conjugation reaction to go at least 4 hours, preferably overnight. (we do 4C overnight) [000197] Example 2: Fabrication of Alginate based aAPCs [000198] Step 1: Preparation of alginate nanoparticles [000199] Materials ^ 5 mL sterile water ^ 100 mg of sodium alginate ^ Sonicator (amplitude 60) ^ Homogenizer ^ 100 mL cyclohexane ^ 1M calcium chloride (CaCL2 in water) ^ 4500mg Span 80 ^ 1500mg Tween 20 [000200] Alginate preparation (water / oil phase / surfactant) Water (Alginate) (for 5mL total): 1. Place 5mL water on 90C hot stir plate to heat up 2. Weigh out 100mg alginate and add (slowly) to the hot 5mL DH20 3. let heat on heated stir plate for ~10 min or until alginate dissolves Oil Phase / Surfactant (nanosized): 1. Weigh out 4500mg of Span80 slowly with a plastic dropper 2. Tare and weigh out 1500mg of Tween80 slowly in the same small beaker 3. Add 100 mL cyclohexane and stir bar 4. Put oil and surfactant solution on stir plate at 500rpm 5. Put oil and surfactant solution on stir plate at 500rpm until completely combined. 6. Remove stir bar. Oil Phase / Surfactant (microsized): 1. Weigh out 1500mg of Span80 slowly with a plastic dropper 2. Tare and weigh out 500mg of Tween80 slowly in the same small beaker 54 162665565.13. Add 100 mL cyclohexane and stir bar 4. Put oil and surfactant solution on stir plate at 500rpm until completely combined. 5. Remove stir bar. [000201] Emulsify and crosslink 1. Place oil and surfactant solution on ice and sonicate for 1 min at 60 amp for nanosized particles. (5000 rpm for micros) 2. Add alginate solution and allow to sonicate for an additional 1 min at 60 amp. (5000 rpm for micros) 3. Add ~3 mLs (3 droppers full) of 1 M CaCl2 solution and continue to sonicate for 2 full minutes. 4. Pour out the particle solution evenly into four 50 mL tubes and top off with water, spin at 100xg to remove any large bits. 5. Slowly pour out the particle mixture into new centrifuge tubes until the line between the particles and the water shows. It is key that you don’t pour in the “line” of particles between the solution and the water. 6. Spin down at 21000xg. 7. Discard the supernatant. 8. Resuspend in ethanol by sonicating at 30 amp. 9. Spin down at 21000xg. 10. Resuspend in DI water and sonicate. Spin down at 21000xg. Repeat once more. 11. Resuspend in 5mL of DI H20. [000202] Sizing 4. place 990uL of DH20 in a zetasizer cuvette 5. add 10uL of particle solution into cuvette and mix 6. measure size 3x on the zetasizer [000203] Finding the concentration 1. Weigh a 500mL eppendorf tube and record the empty weight 2. Mix and pipette 150uL of particle solution into the tube 3. Leave tube open and place carefully in 50mL tube with no lid 4. Wrap a kimwipe across the top of the tube and secure with a rubber band 5. Place in -80C and freeze for 30 min (should be fully frozen) 6. Place in lyophilizer overnight 7. The next day, remove from lyophilizer and record weights of tubes. 8. Back-calculate particle concentration and dilute to 5mg / mL 9. Make 5mg aliquots at 5mg / mL and freeze at -20C [000204] Step 2: Surface protein conjugation for aAPC fabrication 55 162665565.1[000205] Materials ^ Sterile water ^ 2-(N-morpholino)ethanesulfonic acid (MES) ^ MES Buffer ^ 787 mg MES ^ Fill tube up to 40 mL with milliQ water ^ Add 2.5 mL of 1M NaOH. Test the pH using a pH strip and add NaOH in 500 uL increments until the pH is 6.0 ^ Sodium Hydroxide (NaOH) ^ N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) ^ N-hydroxysuccinimide (NHS) ^ Peptide loaded MHC-Ig ^ Anti-CD28 ^ 1X PBS [000206] Protein conjugation 1. Take a 5 mg aliquot, thaw, and spin down at 21000xg. 2. Remove the DI water. 3. Resuspend with 900 uL of MES buffer. 4. Prepare EDC / NHS a. 100 uL of EDC / NHS in MES buffer per 2.5 mgs or particles b. Weigh out the appropriate amounts of EDC and NHS: i. 40 mg EDC and 48 mg NHS per 1 mL of MES buffer 5. Quickly add 100 uL to particles. 6. Put the particles on the inverter at room temperature for 30 minutes. 7. Make the required protein solution: a. 5 ug of each protein per mg of particles in 1X PBS 8. Take tubes off inverter and spin at 21000xg for nanos to remove the supernatant. 9. Resuspend in 1 mL PBS: a. Vortex to resuspend microparticles b. Sonicate with the microtip at 30% amplitude for 5 seconds to resuspend nanoparticles 10. Wash 2 more times with 1X PBS. 11. After the final wash resuspend in 1 mL of 1X PBS. 12. Add protein solution and put on the inverter in the cold room. 13. Allow the conjugation reaction to go at least 4 hours, preferably overnight. (we do 4C overnight) [000207] Example 3: Artificial Antigen Presenting Cell fabrication for Murine T cell Expansion 56 162665565.1[000208] Antigen presenting cells (APCs), such as dendritic cells and macrophages, have a unique ability to survey the body and present information to T cells via peptide loaded major histocompatibility complexes (signal 1). This presentation, along with a co-stimulatory signal (signal 2), leads to the activation and subsequent expansion of T cells. This process can be harnessed and utilized for therapeutic applications; however, the use of patient derived APCs can be complex and inefficient. Alternatively, artificial APCs (aAPCs) provide a simplified method to achieve T cell activation by presenting the two necessary stimulatory signals. This protocol describes the utilization of magnetic nanoparticles and stimulatory proteins to create artificial antigen-presenting cells (aAPCs), which can subsequently be employed for activating and expanding antigen-specific T cells for both basic and translational immunology and immunotherapy studies. Basic Protocol 1: Protein and particle modification for aAPC fabrication Basic Protocol 2: Protein confirmation for aAPC quality check Support Protocol 1: Preparation of particle concentration curve for stock concentration Basic Protocol 3: Determine aAPC use for CD8+ t cell activation Support Protocol 2: CD8+ T cell isolation INTRODUCTION: [000209] T cells play a crucial role within adaptive immunity; aided by professional antigen presenting cells (APCs), such as dendritic cells or macrophages, T cells have the ability to precisely locate and kill diseased host cells. During the direct cell-to-cell interaction between APCs and T cells, APCs present multiple proteins that are designed to stimulate intracellular signaling in T cells that propagate cell activation and proliferation. There are 3 main signals required for successful T cell activation and proliferation: the peptide-loaded major histocompatibility complex (pMHC) (signal 1), costimulatory signal such as CD80, (signal 2), and cytokine support (signal 3)(Curtsinger et al., 1999; Smith-Garvin et al., 2009). All 3 signals help to instruct antigen-specific T cells to promote proper expansion and acquire cell function. Although, unlike cytokine support, which can be both autocrine and paracrine, signals 1 and 2 are solely presented by APCs (Čemerski & Shaw, 2006). 57 162665565.1[000210] Due to their specific role in T cell activation, APCs have been isolated for ex vivo stimulation of T cells in cases of both scientific investigation and in clinical settings for cancer immunotherapies (Chodon et al., 2014; Wölfl & Greenberg, 2014). However, current methods for isolating and preparing APCs from individual patients can lead to increased cost of treatment and are subject to differences in individual cell functionality (Est-Witte et al., 2021). To combat these challenges, artificial APCS (aAPCs) have been developed to mimic the role of APCs by interacting with T cells and presenting signals 1 and 2(Neal et al., 2017). The use of aAPCs, rather than patient derived APCs, allows for more consistent T cell stimulation and greater control over the amount of signal each T cell receives (Almand et al., 2000; Kosmides et al., 2018). [000211] Here we provide protocols for the fabrication and use of aAPCs made from magnetic nano-sized particles. Specifically, Basic protocol 1 describes how to chemically modify both the particles and protein for protein attachment. Basic protocol 2 demonstrates how to validate the presence of protein on the particle surface via secondary antibody staining with Support Protocol 1 detailing how to quantify the concentration of aAPCs post fabrication. Basic protocol 3 describes the general approach towards using aAPCs to activate T cells and establishes a way to identify appropriate dosing of the aAPCs for T cell expansion. Lastly, Support Protocol 2 describes the method for isolation and preparation of murine T cells for aAPC stimulation. While the protocols here describe approaches that are relevant for expansion of murine T cells, similar work has been done with HLA-Ig based aAPCs to expand human T cells (Oelke et al., 2003). [000212] NOTE: All protocols using animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals [000213] BASIC PROTOCOL 1: [000214] PROTEIN AND PARTICLE MODIFICATION FOR aAPC FABRICATION [000215] aAPCs can be prepared with a variety of particle sizes, shapes, materials, and chemistries (Ben-Akiva et al., 2023; Hickey et al., 2020; Rhodes et al., 2021). This protocol outlines how to conjugate peptide loaded MHC-Ig (pMHC-Ig) (Schneck et al., 2001) and anti- CD28 to 250 nm NH2 terminated particles for aAPC fabrication. This protocol may also be used 58 162665565.1to attach other proteins, as it relies on bonding between any thiolated protein and the maleimide groups on the particles post modification, i.e a thiol-maleimide reaction (Northrop et al., 2015). This protocol involves the use of two chemicals, Traut’s reagent and Sulfo-SMCC, that pose risks if inhaled and should be used within a fume hood or in an area equipped with adequate ventilation. If the protocol is done correctly, both proteins will successfully attach to the surface of the particle and a solution of aAPCs, free of aggregation, will be recovered. Materials Peptide loaded MHC-Ig (pMHC-Ig) (see Schneck, 2001) Anti-CD28 (Bio X Cell, BE0015-1) 1X phosphate-buffered saline (PBS, e.g., Corning, 21-040-CM) 1X PBS EDTA buffer (see recipe) Pierce Traut's Reagent [2-iminothiolane] (Thermo Scientific, 26101) Sterile Water (dH2O, e.g Corning, MT46000CI) Conjugation buffer (see recipe) nanomag-D 250 nm magnetic NH2 particles (Micromod, 09-01-252) 10X PBS EDTA buffer (see recipe) Sulfo-SMCC (Thermo Fisher Scientific, 22322) L-Cysteine (Thermo Fisher Scientific, A10435-30) Biosafety Cabinet (e.g., Labconco Purifier BSC Class II) 1.7 mL microcentrifuge tubes (e.g VWR, 87003-294) Pipettes Lab balance (e.g Mettler Toledo, 30315631) Fume hood (e.g Labflex Omega 2600) Aluminum Foil Tube Rotator (e.g Fisher Scientific, 88-861-051) Amicon Ultra-0.5 Centrifugal Filter Unit (Millipore sigma, UFC5050) Benchtop centrifuge (e.g Fisher Scientific, 75002541) Spectrophotometer (e.g Thermo Fisher Scientific, ND-ONE-W) Vortex mixer (e.g Corning, 6775) Fisherbrand 13 mm Glass Screw Thread Vials (Fisher Scientific, 03-391-37) EasyEights™ EasySep™ Magnet (StemCell Technologies, 18103) Protein thiolation / modification 1. Calculate the individual amount of pMHC-Ig and anti-CD28 needed based on the amount of particles being used. ^^ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^  ൌ ^^^ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  ൈ  0.05  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^^  ൈ   ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^ ^^ ^^ ^^ℎ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^ ^^ ^^ ^^ℎ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^ ^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^59 162665565.1We recommend starting with 2 mgs of particles for each kind of aAPC made along with 0.1 mg of total protein (protein A + protein B). An estimated molecular weight of 150,000 Da and 250,000 Da can be used for antibodies and pMHC-Igs, respectively. In the case where anti-CD3 is used instead of a pMHC-Ig, equal mg amounts of signal 1 and signal 2 can be used. 2. Place both proteins in a sterile biosafety cabinet. 3. Label two 1.7 mL microcentrifuge tubes for the pMHC-Ig and anti-CD28 so that the proteins are kept separate. Pipette the calculated amount of each, from their stock, into their respective tube. 4. Dilute each protein, using 1X PBS, to a concentration of 1 mg / mL. The 1 mg / mL stock is made in 1X PBS for the sake of this procedure but is not recommended for long term storage. Therefore, we advise making a small stock at this concentration and following the manufacturer’s protocol for long term storage of the remaining protein stock. 5. To each tube add 1X PBS EDTA buffer to reach a final concentration of 20 mM EDTA. 6. Prepare a 1 mg / mL solution of Traut’s reagent by weighing out 0.5 - 1 mg of Traut’s reagent in a fume hood and dissolving in sterile water. Traut’s reagent is considered toxic and should be weighed out in a fume hood to prevent inhalation. In addition, any waste should be discarded in an approved waste container. 7. Add 20-fold molar excess of Traut’s reagent to each protein and cover with foil. ^^ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ᇱ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^ ^^  ^^ ^^ ^^  ൌ   ^^ ^^  ^^ ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^  ൈ  ^^ ^^ ^^ ^^ ^^ᇱ^^  ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^  ^^  ^^ ^^  ൈ  20The molecular weight of Traut’s reagent is 137.63 g / mol. 8. Rotate the proteins for 2 hours at room temperature. Around the 1-hour mark it is important to start the modification process for the particles (particle preparation). This allows for both processes, the protein and particle modification, to finish around the same time. 9. After two hours, prime the 50-kDa MWCO filter with Conjugation buffer and centrifuge at 14,000xg for 2 min. Discard the flow through. 10. Add the protein to the filter (up to 500 µL at a time) and centrifuge at 14,000xg for 5 min or until the liquid level reduces to 200 µL. Discard this first flow through in an appropriate waste container as it contains traut’s reagent. 11. Wash the protein by adding the Conjugation buffer to the filter and centrifuging at 14,000xg for 5 min or until the sample reaches 200 µL. Discard the flow through. 12. Repeat step 11 two more times for a total of three washes. 13. Add 300ul of Conjugation buffer to protein filter and pipette gently to mix. 14. Place the filter upside down into a fresh tube. 15. Centrifuge the tube with the filter still inside at 1,000xg for 2 min to collect the protein. 60 162665565.116. Remove the filter. 17. Measure the concentration of the protein with a spectrophotometer and take note of the final protein amount. Be sure to “blank” the spectrophotometer with the Conjugation buffer prior to measuring your sample. Particle preparation Note that this process begins after the protein has been rotating for 1 hour. The two steps overlap. 1. Vortex or invert the stock tube of particles. 2. Pipette the desired volume of particles into a glass vial. The particles typically come at a concentration of 10 mg / mL, double check the stock concentration to determine how much volume to use. 3. Add 10X PBS-EDTA to reach 0.01 M EDTA. 4. Calculate and weigh out 0.7 µmol of Sulfo-SMCC per mg of particles. Be sure to measure out Sulfo-SMCC in a fume hood and cover with foil. When weighing out Sulfo-SMCC it is not necessary to be exact, weighing out a little more than you need is advised. 5. Resuspend the Sulfo-SMCC in sterile water at a concentration of 10 mg / mL and vortex for 1 full minute. Then add 0.7 µmol Sulfo-SMCC per mg of particles to the glass vial and cover with foil. 6. Rotate for 1 to 1.5 hours at room temperature. 7. Wash the sample on a magnetic column by resting the glass vial against the magnet and waiting 1 minute. The liquid will become clearer, and you should see the particles sticking at the back of the vial towards the magnet. 8. Press the vial against the magnet to stabilize it and then remove the liquid. Be sure not to touch the magnetic particles on the side of the vial during removal. 9. Remove the vial from the magnet, add 500 µL of diluted 10X PBS-EDTA buffer (1:10 in sterile water) for every 2 mgs of particles and vortex until all the particles are resuspended. 10. Repeat steps 7-9 two more times. After repetition of the steps, you should have your particles in 500 µL of diluted 10X PBS-EDTA per 2 mgs of particles. We recommend having 500 µL of volume for every 2 mgs of particles to ensure they are diluted, however this is not mandatory. 61 162665565.1Protein and particle combination Once both the protein and particles have been modified, they may be combined to form aAPCs. 1. Combine the proteins with the particles, vortex, and let rotate overnight at room temperature to form aAPCs. 2. Quench the reaction the next morning by adding up to 4000 nmols of L-cysteine in 1X PBS. 4000 nmols of cysteine can be used to quench up to 25 mgs of aAPCs post reaction. 3. Rotate for 1 hour at room temperature. 4. Wash the aAPCs on the magnetic column with 1X PBS. Repeat for a total of three washes. 5. Resuspend the aAPCs in 500 µL of 1X PBS for every 2 mgs of aAPCs. BASIC PROTOCOL 2: PROTEIN CONFIRMATION FOR aAPC QUALITY CHECK [000216] aAPC quality is primarily dictated by the conservation of protein functionality and the efficiency of protein conjugation. This protocol details how to verify effective protein binding of pMHC-Ig and anti-CD28 on 250 nm NH2 terminated particles. It may also be used to assess other proteins conjugated to the surface of the aAPC. This protocol is written in such a way that all of the calculations can be performed individually by hand, however, we strongly recommend using a spreadsheet application such as Microsoft Excel in order to reduce both time and potential calculation errors. We have provided an example plate diagram, which may be used as a guide during the protocol. If you decide to alter the plate layout, it is important that you adjust accordingly. After completion you will be able to confirm the successful fabrication of the aAPCs made in Basic Protocol 1. Materials aAPCs (see Basic Protocol 1) 1X phosphate-buffered saline (PBS e.g., Corning, 21-040-CM) Goat anti-Syrian Hamster IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, A21451) Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A11001) Biosafety Cabinet (e.g., Labconco Purifier BSC Class II) Pipettes Fisherbrand 13 mm Glass Screw Thread Vials (Fisher Scientific, 03-391-37) Aluminum Foil 62 162665565.11.7 mL microcentrifuge tubes (e.g VWR, 87003-294) Corning 96 Half Area Well Solid Black Flat Bottom Polystyrene TC-treated Microplate (Corning, 3875) EasyEights EasySep Magnet (StemCell Technologies, 18103) Plate Reader (e.g Thermo Fisher Scientific, VL0000D0) aAPC Sample Preparation 1. Pipette 25 µL of the aAPC stock (Basic Protocol 1) into a glass vial while in a sterile hood. This sample will be used for staining the conjugated proteins. We recommend keeping the stock of aAPCs sterile, however the sample used for staining can be handled in a non-sterile manner. 2. Create a 1:4 dilution of this sample using 1X PBS. 3. Stain the aAPC sample with 0.5 µL of each fluorescent antibody. This protocol utilizes two antibodies with different fluorophores, therefore both antibodies are added to the same sample aAPC solution. Goat anti-Syrian Hamster IgG (H+L) and Goat anti- Mouse IgG (H+L) are used to identify anti-CD28 and pMHC-Ig, respectively. If using two antibodies with the same fluorophore, only one antibody should be added to the aAPC sample. A separate sample will need to be used to stain the second protein being used. 4. Cover the sample with foil and allow it to stain for 1 hour at 4°C. During this 1 hour waiting period it is helpful, although not mandatory, to prepare your plate for the antibody concentration curve. antibody concentration curve plate setup 5. Pipette 2 µL of the first fluorescent antibody, either for the pMHC-Ig or anti-CD28, into a 1.7 mL microcentrifuge tube. 6. Repeat step 5 for the second fluorescent antibody. In the end there should be two tubes with 2 µL of either fluorescent antibody, but not both combined. 7. Utilizing the same two tubes in step 5 and 6, add 398 µL of 1X PBS. This will make 400 µLs of a 1:200 diluted sample for each fluorescent antibody. These will be used to start the titrations for the antibody concentration curve. 8. Pipette half (200 µL) of one of the diluted antibodies into the first well of the first row of a black 96-well half area flat bottom plate. Then take the remaining half and add it to the first well in the second row. 9. Follow the same process for the other antibody, using the third and fourth rows. Ideally, there should now be 200 µL of the 2 diluted antibody samples in wells A1, B1, C1, and D1. 10. Add half the amount of 1X PBS (100 µL) to wells 2-12 for rows A-D. 11. Begin the titration by transferring half of the volume from well 1 to well 2 for each row and mixing. 63 162665565.1This process will be made easier with a multichannel pipette, although it is not required. 12. After sufficient mixing, take half the solution from well 2 and transfer to well 3 for each row. 13. Continue this process until well 11 has been mixed with antibody in each row. 14. Discard the remaining half from well 11, leaving 100 µL behind after mixing, to keep well 12 as undiluted 1X PBS. Note that at the end of this process every well should have the same amount of solution. 15. Wrap the plate in foil. If this step is done during the 1 hour aAPC staining period, the plate can be placed at 4°C. 16. Remove the stained aAPC samples from the fridge. 17. Place the vials upright on the magnet and wait 1 minute. Remove the liquid by pipetting and discard it. Be sure not to touch the magnetic particles on the side of the vial. Although there are less aAPCs in the stained samples, you should be able to see the aAPCs move to the side of the vial towards the magnet (usually in a vertical line). The remaining solution may or may not be clear after this period of time based on how well the aAPCs are drawn to the magnet. 18. Remove the vial from the magnet and resuspend the aAPCs in 500 µL of 1X PBS and vortex. 19. Repeat steps 17 and 18 two more times for a total of three washes. 20. Transfer 200 µL of the sample aAPC solution to the first well of two sequential empty rows of the 96 well plate, e.g. E1 and F1. The remaining 100 µL of sample aAPC solution will be used for particle stock calibration (see Support Protocol 1). 21. Add 100 µL of 1X PBS to wells 2-4 for each row. For example, if the aAPC sample is in E1, add 1X PBS to E2, E3, and E4. 22. Create a titration by transferring 100 µL of the sample aAPC solution from well 1 to well 2 for each row and mix. 23. After sufficient mixing, take 100 µL from well 2 and transfer to well 3. Repeat with well 3 to well 4. Use a plate reader to measure fluorescence in each well. We advise taking multiple measurements of each well (at least three) so that you can calculate the average to reduce the impact of any machine errors. 24. Calculate the average fluorescence for each well in the antibody titrations (rows A-D) as well as the aAPC titrations (rows E and F), excluding the wells containing undiluted 1X PBS. For example, if the fluorescence in well A1 was measured three times and reported as 35.4, 35.2, and 34.7, the calculation for well A1 would be:35.4 ^ 35.2 ^ 34.73 ൌ 35.164 162665565.1After the average is calculated for each well, the averages will be used in the next step. If only one set of measurements was taken, then those directly measured values will be used in the next step. 25. Add the average values for each duplicate well, e.g. A1 and B1 or E1 and F1 and take the average. For the antibody titrations there are 11 samples with duplicate wells while the aAPC titration has 4 samples with duplicates. 26. Find the average background fluorescence value from the wells containing undiluted 1X PBS. 27. Subtract the average background fluorescence value from the averages found in step 26. 28. Multiply all averages from the sample aAPC titration by the amount they were diluted by. For example, if the averages were 2.03, 1.14, 0.597, and 0.292, the calculations would be:2.03 ⋅ 1 ൌ 2.031.14 ⋅ 2 ൌ 2.280.597 ⋅ 4 ൌ 2.390.292 ⋅ 8 ൌ 2.3429. Average the values together to have one final sample aAPC solution fluorescence value. For example:2.03 ^ 2.28 ^ 2.39 ^ 2.344 ൌ 2.26SUPPORT PROTOCOL 1 PREPARATION OF PARTICLE CONCENTRATION CURVE FOR STOCK CONCENTRATION Here we describe methods to calculate the concentration of aAPCs that were fabricated in Basic Protocol 1. This information can be used to guide future T cell activation experiments in which more accurate dosing of aAPCs is necessary, such as dosing by protein amount. Materials Sample aAPCs (see Basic Protocol 2) aAPCs (see Basic Protocol 1) 1X phosphate-buffered saline (PBS e.g., Corning, 21-040-CM) nanomag-D 250 nm magnetic NH2 particles (Micromod, 09-01-252) Pipettes 96-well Clear Flat Bottom Microplate (e.g., Corning, 353072) Spectrophotometer (e.g. Beckman Coulter AD340 Plate Reader) Graphing program (e.g Excel or Graphpad Prism) Particle Stock Calibration 65 162665565.11. Add the remaining 100 µL of sample aAPC solution (see Basic Protocol 2, step 20) to well A1 in a clear 96 well flat bottom plate. 2. Create a 1:10 dilution of the aAPC stock (see Basic Protocol 1) in 1X PBS and add 100 µL to A2. If using our general recommended amounts, you would add 10 µL of aAPC stock to 90 µL of PBS. 3. Prepare a solution of unmodified particles at a concentration of 0.5 mg / ml in 1X PBS to create a reference particle solution with a known concentration. We suggest making 250 µL of particles at this concentration. 4. Add 200 µL of reference particles to well B1 to start the nanoparticle titration (Table 7) 5. Add 100 µL of 1X PBS to wells B2-B8. 6. Add 100 µL of the reference particles from well B1 to B2 and mix thoroughly. 7. Transfer half of the volume from well B2 to well B3 and mix thoroughly. 8. After sufficient mixing transfer half of the volume from well B3 to well B4. 9. Continue this process to create a serial dilution until well B7 has been mixed thoroughly. 10. After mixing well B7 thoroughly, throw out half of the solution. Well B8 should have undiluted PBS. 11. Use a spectrophotometer with a filter of 405 nm to measure the absorbance in each well. We advise taking multiple measurements of each well (at least three) so that you can calculate the average to reduce the impact of any machine errors. Table 7: Nanoparticle titration set up for concentration curve Well B1 B2 B3 B4 B5 B6 B7 B812. Plot a graph of the concentration (y axis) vs absorbance (x axis) and find the equation of the line. 13. It is helpful to double check your R2value to ensure that the titration was done correctly. 13. Calculate the concentration (y value) of aAPCs in well A1 (sample aAPC) and A2 (stock aAPCs). In order to find the concentration of the original aAPC stock, multiply the concentration by 10 to account for the dilution. BASIC PROTOCOL 3 DETERMINING aAPC USE FOR MURINE CD8+ T CELL ACTIVATION CD8+T cells from different types of mice, transgenic models and wild type models, will respond differently to varying activation levels as they are engineered with different TCRs (Abad et al., 2008; Clarke et al., 2000). Therefore, aAPCs usage will be determined based on the CD8+ T cell 66 162665565.1origin as well as the goal of the study. This protocol outlines a general guide for activation and expansion of CD8+ T cells which will serve as an aid for experimental design. The example shown is for the expansion of transgenic T cells, however there are other resources that establish how to use for expansion from a diverse repertoire of T cells such as wild type b6 mice (Perica et al., 2015). Materials: CD8+ T cells (See support protocol 2) Vybrant™ CFDA SE Cell Tracer Kit (Thermofisher, V12883) M media (see recipe) B media (see recipe) Recombinant Murine IL-2 (Peprotech, 212-12) aAPCs (see basic protocol 1) FACS wash buffer (see recipe) APC anti-mouse CD8a Antibody clone 53-6.7 (e.g Biolgend, 100711) Fixable violet live dead (Thermo Fisher Scientific, L34955) 1X phosphate-buffered saline (PBS e.g., Corning, 21-040-CM) Biosafety Cabinet (e.g., Labconco Purifier BSC Class II) Pipettes 50 mL conical tube (e.g Falcon, 352070) Refrigerated benchtop centrifuge (e.g Thermo Fisher Scientific, 75004521) 1.7 mL microcentrifuge tubes (e.g VWR, 87003-294) CO2 Incubator (e.g Thermo Fisher Scientific, 3110) Cell counter (e.g Hemocytometer) Sterile Water (dH2O, e.g Corning, MT46000CI) 8-Channel Reservoir (e.g Corning, 4878) 96-well u-bottom plate (e.g Corning, 3799) FACS tubes (5 mL u-bottom polysterene tubes, eg Falcon, 352052) FlowJo Software with License Flow cytometer (e.g Attune NxT Flow Cytometer) CFDA-SE Labelling and Fold expansion for proliferation Everything should be done in a sterile hood. 1. Pipette the equivalent of 3 million CD8+ T cells into a 50 mL conical tube. 2. Centrifuge the sample at 1500 rpm for 5 min at room temperature. Discard the supernatant. 3. Make the CFDA-SE solution in a 1.7 mL microcentrifuge tube by adding 1 µL of CFDA- SE to 1 mL of warm M media. We recommend using 1 µL of CFSA-DA for every 3 million cells. 67 162665565.14. Resuspend the CD8+ T cells in 1 mL of the prepared CFDA-SE solution. 5. Incubate the cells at 37 C and 5% CO2 for 20 mins. 6. Add 15 mLs of B media and centrifuge the sample at 1500 rpm for 5 min at room temperature. Discard the supernatant. Although we recommended adding 15 mLs of B media to the sample, it is more important that the CFDA-SE be diluted at least 4X with B media to ensure that it is washed out prior to plating the cells. 7. Resuspend in 5 mL of B media and count to determine the concentration of the cell sample. 8. Resuspend at 200,000 cells per mL of B media. 9. Set aside 200,000 cells to be analyzed by flow cytometry as a Day 0 sample. This will be helpful for setting a baseline. After CFDA-SE staining we do not recommend placing the sample on ice. Keep the cell sample at room temperature or in the incubator until it is time to plate. 10. Make a 20 µg / mL stock solution of IL-2 in B media (2X IL-2 feed) and sterile filter. This will be used to provide initial cytokine support to the cells as well as for feeding the cells on day 3. 11. Add equal volume of the IL-2 to the cells to bring the concentration of the cells to 100,000 cells / mL and the IL-2 concentration to 10 µg / mL. 12. In a 8-well multichannel basin add 2 mLs of the cell solution to 6 wells. 13. Add 16 µL of the aAPCs to the first well, 12 µL to the second, and 8, 4, and 2 µL to the following three rows. The sixth row will be left without any aAPCs and will be an unstimulated control. The samples will include 0, 1, 2, 4, 6, and 8 uL of aAPCs per 100,000 cells. 14. Pipette 160 µL of each sample into the 96-well u-bottom plate. Each sample should have a total of 12 separate wells. 15. Transfer the cells that were set aside (step 9) to a FACS tube, add FACS wash buffer, and centrifuge at 1500 rpm for 5 mins. Discard the supernatant. 16. Stain with 1 µL of anti-mouse CD8a APC and 0.1 µL of fixable violet live dead stain for 15 mins at 4°C. 17. Wash the samples by adding FACS wash buffer and centrifuge at 1500 rpm for 5 mins. 18. Resuspend in 200 µL of PBS and run on the flow cytometer. 19. Place the plate into the incubator at 37°C and 5% CO2 for 3 days. 20. Remove 6 wells from each sample and transfer to FACS tubes. Feed the remaining 6 wells with 80 µL of 2X IL-2 in feed and place the plate back into the incubator for another 4 days. 68 162665565.121. Centrifuge the samples in FACS tubes at 1500 rpm for 5 mins and discard the supernatant. 22. Add FACS wash buffer to the tubes and centrifuge again at 1500 rpm for 5 mins. Discard the supernatant. 23. Prepare a staining master mix with 1 µL of anti-mouse CD8a APC and 0.1 µL of fixable violet live dead stain for every 100 µL of FACs wash buffer. Stain each sample with 100 µL of the mix for 15 mins at 4°C. 24. Add 2-3 mLs of 1X PBS to each FACs tube and centrifuge at 1500 rpm for 5 mins. 25. Resuspend in 200 µL of PBS and run the samples on a flow cytometer. 26. Utilize the FlowJo software to compare the CFDA-SE and live / dead for all the samples including Day 0. 27. Remove the remaining cells from the incubator on day 7 and transfer each sample into a FACS tubes. 28. Centrifuge the samples at 1500 rpm for 5 mins and discard the supernatant. 29. Add PBS to the tubes and centrifuge again at 1500 rpm for 5 mins. Discard the supernatant. 30. Bring each sample up to 500 µL and count to determine the total cell number. 31. Divide the number by the starting cell number to obtain the fold expansion. SUPPORT PROTOCOL 2: MURINE CD8 T CELL ISOLATION Materials: Mouse (ages 8 to 12 weeks) 1X phosphate-buffered saline (PBS e.g., Corning, 21-040-CM) Bleach autoMACS Running Buffer (Miltenyi Biotec, 130-091-221) CD8a+ T Cell Isolation Kit, mouse (Miltenyi Biotec, 130-104-075) B media (see recipe) 50 mL conical tube (e.g Falcon, 352070) 70 µm cell strainers (e.g Falcon, 352350) 1 mL syringe (plunger) (e.g Fisher Scientific, 14955456) Transfer pipette (e.g Fisher Scientific, 13-711-5AM) MACS Separator (Miltenyi Biotec, 130-042-302) MACS Multistand (Miltenyi Biotec, 130-042-303) LS columns (Miltenyi Biotec, 130-042-401 Note that equipment for mouse euthanasia is also necessary. 69 162665565.11. Euthanize the mouse by CO2 inhalation followed by cervical dislocation. 2. Isolate the lymph nodes and spleen and immediately transfer to a 50 mL conical tube with 5 mLs of cold 1X PBS. We recommend isolating the lymph nodes first followed by the spleen to ensure that the lymph nodes can be viewed. In addition, after lymph nodes and spleens are harvested, the remaining steps should be done in a sterile hood. 3. Put the sample on ice and transfer to a sterile hood. 4. Place a 70 µm sterile filter on a 50 mL conical tube. 5. Transfer the spleen and lymph nodes onto the filter. 6. Use the flat side of a syringe plunger to mash the organs through the filter. While mashing, use a transfer pipette to wash your sample with PBS to ensure the contents pass through. You will know that the organs have been processed fully and the cells have passed through once the tissue that remains changes color and becomes pale and white. Also, the final volume should be between 20 and 25 mLs of PBS to ensure the filter is washed properly. 7. Centrifuge at 1500 rpm for 5 min at 4°C. Remove the supernatant and follow it with bleach. 8. Resuspend the pellet in 1 mL of ACK lysing buffer and put on ice. Let it stand for exactly 1 minute then add 1x PBS up to 50 mLs. Centrifuge 5 mins at 1500 rpm, 4°C. ACK is toxic to cells so it is important that its left for no more than 1 minute. 9. Resuspend in 10 mLs PBS and put through a second cell strainer on a new 50 mL conical tube. Take an additional 10 mLs of PBS and wash the original tube, pass through the cell strainer. 10. Count the cells. 11. Centrifuge at 1500 rpm for 5 min at 4°C. Dump the supernatant and remove liquid on edge of conical tube before turning upright. This is to reduce the amount of PBS in the sample to avoid diluting the AutoMacs Running buffer. 12. Resuspend the pellet in 40 µL of AutoMacs Running buffer for every 107cells. 13. Utilize the mouse CD8+ isolation kit and add 10 µL of solution 1 (biotinylated antibodies) for every 107cells. Incubate in the fridge for 5 minutes. 70 162665565.114. Add an additional 30 µL of AutoMacs Running buffer for every 107cells. 15. Add 20 µL of solution 2 (anti-biotin beads) for every 107cells. Incubate for 10 minutes. 16. During the 10 minute incubation period, place the MACS Separator onto the multistand and then place a LS Column onto the Macs separator. 17. Put a 50 mL conical tube underneath it to collect the flow through. The CD8 kit is a “no touch” kit, meaning that the desired cells will pass through and will not be bound to the magnetic column. 18. Wet the column with 3 mLs of AutoMacs Running buffer and allow it to fully pass through. 19. Add the cell sample to the column and allow to pass through completely. 20. Wash the tube with the residual sample with 4 mLs of AutoMacs Running buffer and add it to the column. 21. Add B media to the cell sample to bring the total volume up to 40 mLs. 22. Centrifuge the tube at 1500 rpm for 5 min at 4°C. 23. Resuspend the cells in 10 mLs of B media and count. REAGENTS AND SOLUTIONS: 1X PBS EDTA Buffer (pH 7.4) (sterile filtered) ^ 1X PBS (e.g., Corning, 21-040-CM) ^ EDTA (0.1 M) (e.g, Sigma Aldrich, E9884) ^ Store up to 1 year at 4°C Conjugation Buffer (pH 6.7) (sterile filtered) ^ 1X PBS (e.g., Corning, 21-040-CM) ^ HEPES Buffer (50 mM) (Corning, 25060-Cl) ^ EDTA (10 mM) (e.g, Sigma Aldrich, E9884) ^ Store up to 1 year at 4°C 10X PBS EDTA Buffer (pH 7.4) (sterile filtered) ^ 10X PBS (e.g., Corning, 21-040-CM) ^ EDTA (0.1 M) (e.g, Sigma Aldrich, E9884) ^ Store up to 1 year at 4°C 71 162665565.1M media (sterile filtered) ^ RPMI 1640 + glutamine (Gibco, 11879-020) ^ 0.1% Ciprofloxacin (10 mg / ml) (Sigma aldrich, 17850) ^ 0.2% MEM B-Mercaptoethanol Solution 1000x (Gibco, 21985-023) ^ 0.4% MEM Vitamin Solution 100x (Gibco, 11-120-052) ^ HEPES Buffer (1M) (Corning, 25060-Cl) ^ MEM Sodium Pyruvate (1mM) (Corning, 25-000-CI) ^ MEM Non-essential Amino Acid Solution (0.1 mM) (Corning, 25-025-CI) ^ store up to 3 months at 4°C B media (sterile filtered) ^ RPMI 1640 + glutamine (Gibco, 11879-020) ^ 10% FBS (Gibco, 10438-026) ^ 0.1% Ciprofloxacin (10 mg / ml) (Sigma aldrich, 17850) ^ 0.2% MEM B-Mercaptoethanol Solution 1000x (Gibco, 21985-023) ^ 0.4% MEM Vitamin Solution 100x (Gibco, 11-120-052) ^ HEPES Buffer (1M) (Corning, 25060-Cl) ^ MEM Sodium Pyruvate (1mM) (Corning, 25-000-CI) ^ MEM Non-essential Amino Acid Solution (0.1 mM) (Corning, 25-025-CI) ^ store up to 3 months at 4°C FACS wash buffer (sterile filtered) ^ 10% 10X PBS (e.g., Corning, 21-040-CM) ^ 0.05% Sodium Azide ^ 2% FBS ^ Distilled water ^ store up to 1 month at 4°C COMMENTARY: Background information: [000217] The first attempt to activate T cells using artificial platforms occurred 45 years ago (Engelhard et al., 1978; Herrmann & Mescher, 1981). Since then, different systems have been employed using a range of materials, sizes, shapes, and modalities that have come with varying degrees of success (Isser et al., 2021). One of the more popular methods used today are T activator CD3 / CD28 Dynabeads (Neurauter et al., 2007). These magnetic particles are 4.5 um in diameter and are covalently bound to anti-CD3 and anti-CD28 antibodies (“signal 1” and “signal 72 162665565.12” respectively) for T-cell stimulation. Dynabeads have multiple advantages: 1) they can be easily removed from the cellular solution through magnetic separation, 2) beads for stimulation of both mouse and human cells exist allowing for translation from murine to human models, and 3) they work such that the T-cells maintain their in vivo-like function with the proper cytokine profile. [000218] In contrast to Dynabeads, which stimulate T cells non-specifically, the protocol outlined here offers an approach to stimulate T cells in an antigen specific manner. Our aAPCs are customizable, allowing researchers to have control over which stimulatory molecules are bound to the beads. This offers greater specificity over the experimental conditions and even allows for T cell stimulation using specific binding receptors as opposed to the non-specific anti- CD3 stimulation. This is advantageous when trying to stimulate a specific T cell repertoire from an otherwise heterogeneous population. This adaptability becomes increasingly important as cancer research is translated from murine to human models. HLA-Ig based aAPCs have been fabricated using similar methods and can be used for stimulating autologous T-cells that are specific for human cancers (Oelke et al., 2003). [000219] Parameters: [000220] Researchers should be properly trained in animal handling, euthanasia, and dissection prior to attempting this protocol. Performing the spleen and lymph node dissections is a time sensitive task and any tissues removed from the mouse should be put on ice until the researcher is ready to start dissociating the tissues. This will reduce the amount of cellular death after tissues are removed from the body and increase the number of viable T cells that can be isolated. Researchers should also be familiar with cellular staining, flow cytometry techniques, and flow analysis software such as FlowJo. [000221] It is important that researchers pay particular attention to parts of the protocols that are done “under a laminar hood” or "sterilely” as the components being used at those times are susceptible to contamination when out in the open air. Additionally, when vials and plates are "wrapped in foil” this is due to light sensitivity and special attention should be given to this instruction. Troubleshooting: Table 8. Troubleshooting Guide for aAPC fabrication and use Problem Possible Cause Solution73 162665565.1Excessive protein loss Increased sample temperature due When washing the protein after to overheated minicentrifuge modification, use a centrifuge that n e e. C t a e r mUnderstanding Results: [000222] Following completion of Basic Protocol 1, you will have conjugated pMHC-Ig and anti-CD28 antibodies to your magnetic nano-sized particles. The final particle solution may be lighter in color than the original unmodified stock as the concentration of particles has been 74 162665565.1reduced. Verification of successful particle-protein conjugation can be performed by following Basic Protocol 2. [000223] At the end of Basic Protocol 2 you will use a plate reader to take measurements of your antibody and sample aAPC titrations. These measurements will show the amount of antibody bound to the particles. Since the antibodies are specific for the proteins that are supposed to be bound to the particles, their attachment to the particles is confirmation of successful protein- particle conjugation. There should be a steady decrease in fluorescence from left to right as you dilute the antibodies in half each time. If the fluorescence from the sample aAPC solution is greater than the “background” fluorescence of PBS, this confirms the presence of protein conjugated to the particles. The value should also fall between the lowest and highest fluorescence values measured in your antibody dilution. [000224] In Basic Protocol 3, the aAPCs are incubated with CD8+ T cells to determine how the cells may react to different levels of stimulation. On day 3 of T cell expansion, CFDA-SE labelled cells are examined to identify changes in the intensity of the dye. This directly correlates to the proliferation of the cells. A bright dye represents lower proliferation and as the cells begin to divide the overall brightness decreases. In addition to CFDA-SE, one can determine the fold proliferation on day 7 to determine how well the proliferated given the different amounts of aAPCs given. In the example data, the 2C CD8+ T cells have a high affinity for their cognate antigen and in this case, the group with the least aAPCs resulted in a similar CFDA-SE dilution to the group with the most aAPCs. However, on day 7 in the example shown we may have reached a plateau or overstimulated the T cells. This can be further controlled by limiting aAPC stimulation or adding additional feedings to the cultures. We want to reiterate that cells derived from different mouse types will differ in their response and these two readouts should be used as a guide to determine what works best for the particular system being used. [000225] Time Considerations: [000226] Basic Protocol 1 – This protocol will take an estimated total time of 4.5 hours with an overnight step. It will take no more than 30 mins to set up, 3 hours for protein thiolation and particle modification, and the aAPC fabrication is an overnight step with an additional 1 hour quench the following day. 75 162665565.1[000227] Basic Protocol 2 – This protocol will take an estimated total time of 2.5 hours.30 mins to set up, 1.5 hour to stain and prepare the samples, and 30 minutes to analyze. [000228] Basic Protocol 3 – This protocol will take an estimated total time of 4 hours.1 hour to isolate the CD8+ T cells, 30 mins to CFDA-SE label them, 30 mins to plate the cells, 1 hour to harvest and perform flow cytometry on day 3, and 1 hour to harvest and count the cells on day 7. [000229] Example 4 [000230] MATERIALS AND METHODS [000231] Mice [000232] C57BL / 6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, Michigan, USA) and 2C TCR transgenic mice were gifted from the Gajewski laboratory (The University of Chicago, Chicago, Illinois, USA)

[0024] . A colony of 2C TCR transgenic mice was maintained by breeding the mice as heterozygotes on the C57BL / 6 background. Both male and female mice, 8-12 weeks old, were utilized for this study and maintained in adherence to committee guidelines outlined by the Johns Hopkins University Animal Care and Use Committee (ACUC). [000233] Cells [000234] B16-SIY cells were gifted from the Drake laboratory (JHU, Baltimore, Maryland, USA). All murine T cells were cultured in media (RPMI supplemented with L-glutamine (Gibco, Thermo Fisher, Massachusetts, USA), 1X MEM nonessential amino acid solution (Gibco, Thermo Fisher, Massachusetts, USA), 1mM MEM sodium pyruvate (Gibco, Thermo Fisher, Massachusetts, USA), 0.4X MEM vitamin solution (Gibco, Thermo Fisher, Massachusetts, USA), 92 µM MEM 2- mercaptoethanol solution (Gibco, Thermo Fisher, Massachusetts, USA), 10 µM ciproflaxin, 1M HEPES buffer, and 10% fetal bovine serum) which was supplemented with T cell growth factor cocktail (TF) as described in Oelke et al

[0025] . B16-SIY tumor cells were cultured in media supplemented with 400 μg / mL G418 (Gibco, Thermo Fisher, Massachusetts, USA). [000235] Nanoparticle Fabrication and characterization 76 162665565.1[000236] A water-in-oil nanoemulsion method was utilized for the production of alginate nanoparticles.100 mg of sodium alginate (Sigma Aldrich, St. Louis, Missouri, USA) was dissolved in 5 mLs of deionized water (diH2O) to prepare a 2% alginate solution for the water phase.4500 mg of Span80 (Sigma Aldrich, St. Louis, Missouri, USA), 1500 mg Tween80 (Sigma Aldrich, St. Louis, Missouri, USA), and 100 mL of cyclohexane (Sigma Aldrich, St. Louis, Missouri, USA) were combined for the oil phase. The oil phase was then sonicated for 1 minute using a probe sonicator (Sonics and Materials Inc, Newtown, Connecticut, USA) at 60% amplitude prior to the addition of the alginate solution. The mixture was then sonicated for an additional 2 minutes and 3 mL of 1M calcium chloride was added to crosslink the alginate. To wash the nanoparticles, the solution was distributed into 50 mL conical tubes, topped off with diH2O and centrifuged (100 x g, 5 min). The diH2O was discarded along with any surfactant at the interface of the diH2O and cyclohexane. The remaining sample was then centrifuged (21000 x g, 5 min). The supernatant was discarded and the nanoparticles were washed once more by resuspending the nanoparticles in ethanol and centrifuging at (21000 x g, 5 min). Two more washes were then conducted in diH2O. After the washes were complete, the sample was resuspended in 5 mL of diH2O. A ZetaSizer Pro (Malvern Panalytical Ltd, Malvern, UK) was then utilized to determine particle diameter, polydispersity index, and zeta potential. For further validation of nanoparticle formation and shape, TEM images were acquired via a Hitachi 7600 TEM (Hitachi High-Tech, Tokyo, Japan). To determine the concentration of the nanoparticle solution, the weight of a microcentrifuge tube was recorded prior to the addition of 200 µL of the of the particles. The particles were then frozen and lyophilized. The weight of the tube was then recorded to determine the weight per solution of nanoparticles. The stock of alginate nanoparticles was then diluted to 5 mg / mL, aliquoted in 1 mL stocks, and stored at -20 ºC. [000237] aAPC Fabrication and Characterization [000238] The pMHC-Ig, KbSIY, was produced in the lab as previously described

[0026] . Iron oxide aAPCs, were fabricated from iron oxide beads (Ocean Nanotech, San Diego, California, USA) as previously described [27,28]. To fabricate alginate based aAPCs, either anti-mouse CD3 (Bio X Cell, Lebanon, New Hampshire, USA) and anti-mouse CD28 (Bio X Cell, Lebanon, New Hampshire, USA) or KbSIY and anti-CD28 were conjugated to the alginate nanoparticles via 77 162665565.1EDC / NHS chemistry. Alginate nanoparticles in ddH2O were centrifuged (21000 x g, 5 min) and resuspended at 2.5 mg / mL in 0.1 M MES buffer (pH 6.0) with 100 µL of an EDC / NHS solution (40 mg / mL EDC and 48 mg / mL NHS in MES buffer) and rotated at room temperature for 30 minutes. The nanoparticles were centrifuged (21000 x g, 5 mins) and resuspended in 1X PBS for washing via centrifugation (21000 x g, 5 mins). The nanoparticles were resuspended at 2 mg / mL in 1X PBS and 5 µg of both antibodies per mg of particles were added. The nanoparticles were incubated at 4 ºC for 24-48 hours. The nanoparticles were then washed with 1X PBS, resuspended in B media, and then stored at -20ºC until use. [000239] To quantify the amount of protein conjugated to the alginate-based aAPCs, the aAPCs were stained with fluorescently labelled secondary antibodies against anti-CD3, anti-CD28, or KbSIY; Chicken anti-Rat IgG (Invitrogen, Thermo Fisher, Massachusetts, USA), Goat anti- Syrian Hamster IgG (Invitrogen, Thermo Fisher, Massachusetts, USA), and Goat anti-Mouse IgG (Invitrogen, Thermo Fisher, Massachusetts, USA), respectively. An aliquot of 2 mgs from the aAPC stock was centrifuged (21000 x g, 5 min) and resuspended in 1 mL of 1X PBS. The aAPCs were stained with 5 µg of the secondary antibodies per every mg of aAPCs and rotated at room temperature for 1 hour. aAPCs were then washed with PBS 3 times and measured on a Biotek Synergy 2 Plate Reader (Agilent Technologies, Santa Clara, California, USA) along with a standard secondary antibody curve for each stain. A comparison of the fluorescence of each secondary antibody to its standard curve was used to determine the amount of conjugated protein per 1 mg of aAPCs as well as the conjugation efficiency of the EDC / NHS reaction. To determine the stability of the protein attached, anti-CD3 / anti-CD28 based aAPCs were fabricated and the amount of conjugated protein per mg was determined on day 0. aAPCs in 1X PBS were then incubated at 37°C for 7 days. On day 7, aAPCs were centrifuged (21000 x g, 5 mins) and stained to determine the amount of protein remaining. [000240] T cell isolation [000241] Spleens and lymph nodes from B6 or 2C TCR transgenic mice were processed through a 70-μm cell strainer. CD8+ T cells were then isolated using a mouse specific CD8a+ T Cell Isolation Kit and magnetic columns (Miltenyi Biotech, Auburn, California, USA) following the manufacturer’s instructions. 78 162665565.1[000242] Ex vivo T cell expansion [000243] For ex vivo expansion of isolated 2C TCR CD8+ T cells, cells were plated on day 0 at 100,000 cells / mL with aAPCs with the equivalent of 18 ng KbSIY / mL. Cells were incubated for 7 days and refed on day 3 with media supplemented with TF at half the initial volume and twice the initial concentration of TF, unless otherwise stated. For expansion of antigen specific T cells from B6 CD8+ T cells, cells were plated at 250,000 cells / mL with 9 ng of KbSIY / mL and split and fed on day 3 and day 5 with media supplemented with TF (0.5x volume, 2X TF). [000244] Antigen specific dimer staining [000245] For identification of KbSIY+ CD8+ T cells, cells were stained at for 1 hour at 4°C with 1 µg of biotinylated KbSIY, 1:100 of PerCP anti-mouse CD8 clone 53-6.7, and 1:1000 Fixable Violet Live / Dead (Invitrogen, Thermo Fisher, Massachusetts, USA). The cells were washed twice (1500 rpm, 5 mins) and stained for 30 mins at 4°C with 1:100 FITC anti-mouse / human CD44 clone IM7 (BioLegend, San Diego, California, USA), APC-Cy7 anti-mouse CD62L clone MEL-14 (BioLegend, San Diego, California, USA), and 1:350 PE Streptavidin (BD Biosciences, Franklin Lakes, New Jersey, USA). The cells were then washed, resuspended in 1X PBS, and analyzed via the Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). [000246] ROS study [000247] Tissue culture treated 96-well plates were coated with anti-CD3. Each well was covered with 50 µL of anti-CD3 (10 µg / ml) and placed at 4°C overnight. The following day, each well was washed twice with 1X PBS before use. B6 CD8+ T cells were then plated at 100,000 cells / mL in media supplemented with TF and soluble anti-CD28 at 1 µg / ml and incubated at 37°C and 5% CO2. Samples incubated with alginate nanoparticles received 0.25 mgs / mL. For detection of mitochondrial based ROS, on day 3 cells were refed with media supplemented with TF (0.5x volume, 2X TF) and ROS was induced with 30 µM MitoPQ (Abcam, Boston, Massachusetts, USA) for 18 hours. On day 4 cells were stained with MitoSOX Green Mitochondrial Superoxide Indicator (Invitrogen, Thermo Fisher, Massachusetts, USA), according to the manufacturer’s protocol. To determine activation levels of the B6 CD8+ T cells, on day 7 cells were stained for 20 minutes at 4°C with 1:1000 Fixable Violet Live / Dead (Invitrogen, Thermo Fisher, Massachusetts, USA), 79 162665565.11:100 APC-Cy7 anti-mouse CD8 clone 53-6.7, 1:100 FITC anti-mouse PD-1 clone 29F.1A12, 1:100 PerCP / Cy5.5 anti-mouse / human CD44 clone IM7, 1:100 Brilliant Violet 711 anti-mouse Tim-3 clone RMT3-23, and 1:100 PE / Cy7 anti-mouse LAG-3 clone C9B7W from Biolegend (San Diego, California, USA). [000248] Phenotypic analysis [000249] For transcription factor staining the Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, Thermo Fisher, Massachusetts, USA) was used. Expanded 2C CD8+ T cells were washed by centrifugation (1500 rpm, 5 mins), resuspended in Facs wash buffer, and stained with 1:100 Brilliant Violet 605 anti-mouse CD8 clone 53-6.7 (BioLegend, San Diego, California, USA) and 1:1000 Fixable Near IR Live / Dead (Invitrogen, Thermo Fisher, Massachusetts, USA) for 15 minutes at 4°C. The cells were washed and resuspended in Foxp3 Fixation / Permeabilization solution, fixed, and permeabilized for 1 hour at 4°C. Post incubation, the cells were washed with 1X Permeabilization Buffer and stained with 1:100 PerCP / Cy5.5 anti-mouse Tbet clone 4B10 (BioLegend, San Diego, California, USA), 1:100 Brilliant Violet 421 anti-mouse TCF1 / 7 clone S33-966 (BD Biosciences, Franklin Lakes, New Jersey, USA), 1:100 PE anti-mouse Eomes clone Dan11mag (Invitrogen, USA) for 30 minutes at room temperature. Cells were then washed (1500 rpm, 5 mins), resuspended in 1X PBS, and analyzed via flow cytometry using the Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). To assess the phenotype of expanded 2C CD8+ T cells, cells were stained for 20 minutes at 4°C with 1:1000 Fixable Violet Live / Dead (Invitrogen, Thermo Fisher, Massachusetts, USA), 1:100 PerCP / Cy5.5 anti-mouse / human CD44 clone IM7, 1:100 APC anti-mouse CD62L clone MEL-14, and 1:100 APC-Cy7 anti-mouse CD8 clone 53-6.7 from Biolegend (San Diego, California, USA). Cells were then washed (1500 rpm, 5 mins), resuspended, and analyzed using the Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). CD62L+CD44-, CD62L-CD44+, and CD62L+CD44+ CD8+ T cells were deemed naïve, effector-like, or memory-like respectively. [000250] Functional analysis [000251] For intracellular cytokine staining, expanded 2C CD8+ T cells were washed by centrifugation (1500 rpm, 5 mins) and resuspended in fresh media without TF. Reactivated cells 80 162665565.1received 1X cytokine activation cocktail (BioLegend, San Diego, California, USA) and GolgiPlug (BD Biosciences, Franklin Lakes, New Jersey, USA) while corresponding no stimulation controls received only GolgiPlug. Cells were incubated for 6 hours at 37°C and 5% CO2. The cells were then washed (1500 rpm, 5 mins) and stained for surface markers and viability, 1:100 PerCP anti- mouse CD8 clone 53-6.7 (BioLegend, San Diego, California, USA) and 1:1000 Fixable Aqua Live / Dead (Invitrogen, Thermo Fisher, Massachusetts, USA), for 15 minutes at 4 °C. Cells were then fixed and permeabilized with the Cytofix / Cytoperm Fixation / Permeabilization kit (BD Biosciences, Franklin Lakes, New Jersey, USA), washed twice (1800 rpm, 3 mins), and stained for the following cytokines from Biolegend (San Diego, California, USA) for 1 hour at 4 °C: 1:50 FITC anti-mouse / human Granzyme B clone GB11, 1:100 of APC anti-mouse IFN-γ clone XMG1.2, 1:100 PE anti-mouse IL-2 clone JES6-5H4, and 1:100 PE-Cyanine7 anti-mouse TNF-α clone MP6- XT22. Cells were then washed (1800 rpm, 3 mins) and resuspended in 1X PBS and analyzed via flow cytometry using the Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). [000252] In vivo killing assay [000253] B6 mice received 500 cGy of irradiation to induce lymphopenia 1 day prior to the adoptive transfer of 2C CD8+ T cells

[0029] . On day 0, 1 × 1062C CD8+ T cells expanded with alginate-based aAPCs (18 ng KbSIY / mL) or 1 × 106naïve 2C CD8+ T cells were transferred, via retroorbital injections, to the lymphodepleted mice. Mice also received intraperitoneal injections of 30,000 U IL-2 (Prometheus Labs, San Diego, California, USA). On day 6 and 20, spleens and lymph nodes from B6 mice were isolated, passed through a 70-μm cell strainer, and red blood cells were lysed using ACK lysis buffer (Quality Biological, Gaithersburg, Maryland, USA). Cells were then split into CFSE high and CFSE low groups by staining cells with 0.5 μL CFSE or 0.033 μL per 4x107cells, respectively. The CFSE high group was then loaded with 1 μg of SIY peptide per 1x107cells in media for 1 h at 37 °C and 5% CO2. After incubation, the cells were washed twice in 1X PBS and mixed at a 1:1 ratio with the CFSE low group before retroorbital injections were given. In vivo killing data was acquired the following day (day 6, day 21). Spleens and lymph nodes from each mouse were processed and stained for 20 minutes at 4 °C with 1:1000 Live / Dead Fixable Violet (Invitrogen, Thermo Fisher, Massachusetts, USA), 1:100 PerCP anti-mouse CD8 clone 53- 6.7 (BioLegend, San Diego, California, USA), 1:100 APC anti-mouse CD3 clone 17A2 81 162665565.1(BioLegend, San Diego, California, USA), 1:100 APC / Cy7 anti-mouse / human CD44 clone IM7 (BioLegend, San Diego, California, USA), and 1:350 PE conjugated 1B2 to identify the transferred 2C CD8+ T cells. Cells were then washed, resuspended in 1X PBS, and analyzed via flow cytometry using an Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). Percent killing was determined using the following formula: [1-(CFSE low:CFSE high / control group CFSE high: control group CFSE low)] x100. [000254] Tumor treatment [000255] 2 × 106B16-SIY tumor cells were injected subcutaneously on the right flank of B6 mice. When the tumor was palpable on day 5, the mice received 500 cGy of irradiation. The following day, retroorbital injections of either 1 × 1062C CD8+ T cells expanded with alginate- based aAPCs for 7 days or 1 × 106naïve 2C CD8+ T cells were given. On the day of and the day after adoptive transfer, mice received intraperitoneal injections of 30,000 U IL-2 (Prometheus Labs, San Diego, California, USA) to support that adoptive transfer. Tumor size was measured every 2 to 3 days using a digital caliper and was halted for each mouse when the tumor either reached 200 mm2or became necrotic. At either end point, mice were sacrificed with CO2 asphyxiation and cervical dislocation. [000256] Persistence Study [000257] The persistence of adoptively transferred cells was examined by analyzing the percentage of transferred cells in the blood overtime. Mice received 500 cGy of irradiation in preparation for the adoptive transfer. The next day, B6 mice received 1x1062C CD8+ T cells expanded with alginate or iron oxide aAPCs for 7 days. To obtain blood samples, submandibular bleeding of mice using a lancet was performed on days 5, 8, 14, and day 21. On day 21, spleens and lymph nodes were harvested and processed separately. Red blood cells in the spleens were lysed using ACK lysis buffer. Cells from the spleens and lymph nodes were stained for 20 minutes at 4 °C with 1:1000 Live / Dead Fixable Violet (Invitrogen), 1:100 PerCP anti-mouse CD8 clone 53- 6.7 (BioLegend, San Diego, California, USA), 1:100 PE anti-mouse CD3 clone (BioLegend, San Diego, California, USA), 1:100 APC / Cy7 anti-mouse / human CD44 clone IM7 (BioLegend, San Diego, California, USA), and 1:350 APC conjugated 1B2 to identify the transferred 2C CD8+ T 82 162665565.1cells.Cells were then washed, Resuspended in 1X PBS, and analyzed via flow cytometry using an Attune NxT Flow Cytometer (Thermo Fisher, Massachusetts, USA). [000258] Preventative tumor treatment [000259] One day prior to the adoptive transfer, mice received 500 cGy of irradiation. On the day of the adoptive transfer retroorbital injections of either 1 × 1062C CD8+ T cells expanded with alginate or iron oxide aAPCs for 7 days were given. Intraperitoneal injections of 30,000 U IL-2 (Prometheus Labs, San Diego, California, USA) were given both the day of the transfer and the day after.1 × 106B16-SIY tumor cells were then injected subcutaneously on the right flank of B6 mice 14 days later. Tumor size was measured every 2 to 3 days using a digital caliper and was halted for each mouse when the tumor either reached 200 mm2or became necrotic. At either end point, mice were sacrificed with CO2 asphyxiation and cervical dislocation. [000260] Statistical analysis [000261] The data sets for each experiment were analyzed using the statistical software program Prism 10 (GraphPad Software, Inc., La Jolla, California, USA). Significance for each graph is based on a p value less than 0.05 and all error bars in graphs represent the standard error of the mean (s.e.m.), unless stated to be the standard deviation (s.d). All in vitro cell experiments, n = 3-4 biological replicates. For in vivo experiments, n = 3-5 mice per group. Differences between two groups were assessed by Student’s t-test, differences among multiple groups were assessed by one and two-way ANOVAs with Tukey’s or Sidak’s multiple-comparisons tests, respectively. [000262] RESULTS [000263] Fabrication and characterization of alginate nanoparticles [000264] To determine if alginate could impact T cell ROS production and therefore be useful for aAPC fabrication, we fabricated alginate nanoparticles as shown in Figure 1A. Briefly, sodium alginate was sonicated in a bulk water-in-oil emulsion to create droplets. Droplets were subsequently crosslinked with calcium chloride to produce negatively charged 550 nm sized spherical nanoparticles (Fig. 1B-D). B6 CD8+ T cells were then incubated with alginate 83 162665565.1nanoparticles and assayed for the presence of mitochondrial ROS, see Figure 1E, schematic. Cells that received alginate nanoparticles showed a decrease in mitochondrial ROS as seen in the total percentage (Fig.1F-G) and MFI (Fig.1H) of cells positive for MitoSOX Green, a fluorogenic dye that targets superoxide in mitochondria. While changes in ROS production were seen in T cells, no difference in T cell phenotype related to activation was observed. This was demonstrated by the percentage of T cells that exhibited PD-1, Tim-3, and LAG-3 (Fig. S1A) or CD44 (Fig. S1B). This demonstrated that alginate nanoparticles could be an effective base material for our aAPC fabrication. LITERATURE CITED: Abad, J. D., Wrzensinski, C., Overwijk, W., De Witte, M. A., Jorritsma, A., Hsu, C., Gattinoni, L., Cohen, C. J., Paulos, C. M., Palmer, D. C., Haanen, J. B. A. G., Schumacher, T. N. M., Rosenberg, S. A., Restifo, N. P., & Morgan, R. A. (2008). T-Cell Receptor Gene Therapy of Established Tumors in a Murine Melanoma Model. Journal of Immunotherapy, 31(1), 1–6. https: / / doi.org / 10.1097 / CJI.0b013e31815c193f Almand, B., Resser, J. R., Lindman, B., Nadaf, S., Clark, J. I., Kwon, E. D., Carbone, D. P., & Gabrilovich, D. I. (2000). Clinical Significance of Defective Dendritic Cell Differentiation in Cancer. Clinical Cancer Research, 6(5), 1755–1766. Ben-Akiva, E., Hickey, J. W., Meyer, R. A., Isser, A., Shannon, S. R., Livingston, N. K., Rhodes, K. R., Kosmides, A. K., Warren, T. R., Tzeng, S. Y., Schneck, J. P., & Green, J. J. (2023). Shape matters: Biodegradable anisotropic nanoparticle artificial antigen presenting cells for cancer immunotherapy. Acta Biomaterialia, 160, 187–197. https: / / doi.org / 10.1016 / j.actbio.2023.02.023 Čemerski, S., & Shaw, A. (2006). Immune synapses in T-cell activation. Current Opinion in Immunology, 18(3), 298–304. https: / / doi.org / 10.1016 / j.coi.2006.03.011 Chodon, T., Comin-Anduix, B., Chmielowski, B., Koya, R. C., Wu, Z., Auerbach, M., Ng, C., Avramis, E., Seja, E., Villanueva, A., McCannel, T. A., Ishiyama, A., Czernin, J., Radu, C. G., Wang, X., Gjertson, D. W., Cochran, A. J., Cornetta, K., Wong, D. J. L., … Ribas, A. (2014). Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and 84 162665565.1Dendritic Cell Vaccination in Patients with Metastatic Melanoma. Clinical Cancer Research, 20(9), 2457–2465. https: / / doi.org / 10.1158 / 1078-0432.CCR-13-3017 Clarke, S. Rm., Barnden, M., Kurts, C., Carbone, F. R., Miller, J. F., & Heath, W. R. (2000). Characterization of the ovalbumin-specific TCR transgenic line OT-I: MHC elements for positive and negative selection. Immunology and Cell Biology, 78(2), 110–117. https: / / doi.org / 10.1046 / j.1440-1711.2000.00889.x Curtsinger, J. M., Schmidt, C. S., Mondino, A., Lins, D. C., Kedl, R. M., Jenkins, M. K., & Mescher, M. F. (1999). Inflammatory Cytokines Provide a Third Signal for Activation of Naive CD4+ and CD8+ T Cells. The Journal of Immunology, 162(6), 3256–3262. https: / / doi.org / 10.4049 / jimmunol.162.6.3256 Engelhard, V. H., Strominger, J. L., Mescher, M., & Burakoff, S. (1978). Induction of secondary cytotoxic T lymphocytes by purified HLA-A and HLA-B antigens reconstituted into phospholipid vesicles. Proceedings of the National Academy of Sciences, 75(11), 5688– 5691. https: / / doi.org / 10.1073 / pnas.75.11.5688 Est-Witte, S. E., Livingston, N. K., Omotoso, M. O., Green, J. J., & Schneck, J. P. (2021). Nanoparticles for generating antigen-specific T cells for immunotherapy. Seminars in Immunology, 56, 101541. https: / / doi.org / 10.1016 / j.smim.2021.101541 Herrmann, S. H., & Mescher, M. F. (1981). Secondary cytolytic T lymphocyte stimulation by purified H-2Kk in liposomes. Proceedings of the National Academy of Sciences, 78(4), 2488–2492. https: / / doi.org / 10.1073 / pnas.78.4.2488 Hickey, J. W., Isser, A., Salathe, S. F., Gee, K. M., Hsiao, M.-H., Shaikh, W., Uzoukwu, N. C., Bieler, J. G., Mao, H.-Q., & Schneck, J. P. (2020). Adaptive Nanoparticle Platforms for High Throughput Expansion and Detection of Antigen-Specific T cells. Nano Letters, 20(9), 6289–6298. https: / / doi.org / 10.1021 / acs.nanolett.0c01511 Isser, A., Livingston, N. K., & Schneck, J. P. (2021). Biomaterials to enhance antigen-specific T cell expansion for cancer immunotherapy. Biomaterials, 268, 120584. https: / / doi.org / 10.1016 / j.biomaterials.2020.120584Kosmides, A. K., Necochea, K., Hickey, J. W., & Schneck, J. P. (2018). Separating T Cell Targeting Components onto Magnetically Clustered Nanoparticles Boosts Activation. Nano Letters, 18(3), 1916–1924. https: / / doi.org / 10.1021 / acs.nanolett.7b05284 Neal, L. R., Bailey, S. R., M. H., Haupt, C., Paulos, C. M., & Varela,. Presenting Cells in T Cell-Based Cancer Immunotherapies. Journal of Immunology Research and Therapy, 2(1), 68–79. Neurauter, A. A., Bonyhadi, M., Lien, E., Nøkleby, L., Ruud, E., Camacho, S., & Aarvak, T. (2007). Cell Isolation and Expansion Using Dynabeads ®. In Cell Separation (pp.41–73). Springer Berlin Heidelberg. https: / / doi.org / 10.1007 / 10_2007_072 Northrop, B. H., Frayne, S. H., & Choudhary, U. (2015). Thiol–maleimide “click” chemistry: evaluating the influence of solvent, initiator, and thiol on the reaction mechanism, kinetics, and selectivity. Polymer Chemistry, 6(18), 3415–3430. https: / / doi.org / 10.1039 / C5PY00168D Oelke, M., Maus, M. V., Didiano, D., June, C. H., Mackensen, A., & Schneck, J. P. (2003). Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig–coated artificial antigen-presenting cells. Nature Medicine, 9(5), 619–625. https: / / doi.org / 10.1038 / nm869 Perica, K., Bieler, J. G., Schütz, C., Varela, J. C., Douglass, J., Skora, A., Chiu, Y. L., Oelke, M., Kinzler, K., Zhou, S., Vogelstein, B., & Schneck, J. P. (2015). Enrichment and Expansion with Nanoscale Artificial Antigen Presenting Cells for Adoptive Immunotherapy. ACS Nano, 9(7), 6861–6871. https: / / doi.org / 10.1021 / acsnano.5b02829 Rhodes, K. R., Isser, A., Hickey, J. W., Ben-Akiva, E., Meyer, R. A., Kosmides, A. K., Livingston, N. K., Tzeng, S. Y., Schneck, J. P., & Green, J. J. (2021). Biodegradable Cationic Polymer Blends for Fabrication of Enhanced Artificial Antigen Presenting Cells to Treat Melanoma. ACS Applied Materials & Interfaces, 13(7), 7913–7923. https: / / doi.org / 10.1021 / acsami.0c19955 Schneck, J. P., Slansky, J. E., O’Herrin, S. M., & Greten, T. F. (2001). Monitoring Antigen‐ Immunology, 27(1), 591–619. https: / / doi.org / 10.1146 / annurev.immunol.021908.132706 86 162665565.1Wölfl, M., & Greenberg Specific T Cells Using MHC‐Ig Dimers. Current Protocols in Immunology, 35(1). https: / / doi.org / 10.1002 / 0471142735.im1702s35 Smith-Garvin, J. E., Koretzky, G. A., & Jordan, M. S. (2009). T Cell Activation. Annual Review of , P. D. (2014). Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells. Nature Protocols, 9(4), 950–966. https: / / doi.org / 10.1038 / nprot.2014.064 OTHER EMBODIMENTS [000265] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. [000266] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 87 162665565.1

Claims

What is claimed:

1. An artificial antigen presenting cell (aAPC) wherein the aAPC is composed of an antioxidant biomaterial.

2. The aAPC of claim 1, wherein the aAPC is a nanoparticle.

3. The aAPC of claim 1 or 2, wherein the diameter of the nanoparticle is in a range from about 200 nm to about 1000 nm.

4. The aAPC of claim 4, wherein the diameter of the nanoparticle is in a range from about 250 nm to about 900 nm.

5. The aAPC of claim 4, wherein the diameter of the nanoparticle is in a range from about 400nm to about 800nm.

6. The aAPC of claim 1, wherein the antioxidant biomaterial is derived from plants, animals, algae, fungi, bacteria, synthetic materials or combinations thereof.

7. The aAPC of claim 6, wherein the antioxidant biomaterial is derived from algae in combination with one or more antioxidant biomaterials derived from plants, animals, fungi, bacteria or synthetic materials.

8. The aAPC of claim 6, wherein the antioxidant biomaterial is derived from algae.

9. The aAPC of claim 8, wherein the antioxidant biomaterial derived from algae comprises alginate, agarose, carrageenan, fucoidan or combinations thereof.

10. The aAPC of claim 8, wherein the antioxidant biomaterial derived from algae comprises alginate.

11. The aAPC of any one of claims 1-10, wherein the antioxidant biomaterial modulates reactive oxygen species (ROS) and oxidative stress in vitro or in vivo.

12. The aAPC of any one of claims 1-12, further comprising an antigenic peptide, a major histocompatibility complex (MHC), a T cell activation signal, a T cell stimulatory signal, a costimulatory signal, an inhibitory signal or combinations, thereof expressed on a surface of the nanoparticle. 88 162665565.

113. The aAPC of any one of claims 1-12, wherein the nanoparticle further comprises an encapsulated peptide, a nucleic acid, an adjuvant, a therapeutic agent, a toxin, or combinations thereof.

14. The aAPC of any one of claims 1-13, wherein the aAPC comprises one or more dendritic cell markers.

15. The aAPC of claim 14, wherein the dendritic cell markers comprise BDCA-1, CD8, CD8alpha, CD11b, CD11c, CD103, CD205, MHC Class II, BDCA-2, BDCA-4, CD11clowCD45RA, CD123 ILT-7, MHC Class IIlowTLR7, TLR9 or combinations thereof.

16. The aAPC of any one of claims 1-14, wherein the aAPC comprises one or more macrophage markers.

17. The aAPC of any one of claims 1-14, wherein the macrophage markers comprise M1 macrophage markers, M2 macrophage markers or combinations thereof.

18. The aAPC of claim 17, wherein the M1 macrophage markers comprise CD86, CD80, CD68, MHC class II, IL-1R, TLR2, TLR4, iNOS, SOCS or combinations thereof.

19. The aAPC of claim 17, wherein the M2 macrophage markers comprise CD163, MHC class II, SR, MMR / CD206, CD200R, TGM2, DecoyR, IL-1R II, CD86, CD163, TLR1, TLR8, VEGF or combinations thereof.

20. A pharmaceutical composition comprising an artificial antigen presenting cell (aAPC) of any one of claims 1-19.

21. A method of treating cancer comprising administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) of any one of claims 1-19.

22. A method of treating an infectious disease comprising administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) of any one of claims 1-19.

23. A method of modulating an immune response comprising administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) of any one of claims 1-19. 89 162665565.

124. A method of treating an autoimmune disease comprising administering to a subject a therapeutically effective amount of an artificial antigen presenting cell (aAPC) of any one of claims 1-14.

25. An artificial antigen presenting cell (aAPC) formulated as a biological active material. 90 162665565.1

Images