Intracellular delivery of biomolecules to induce tolerance

Abstract

The present invention provides methods for inducing tolerance to an antigen and / or suppressing an immune response to the antigen by passing a cell suspension containing anucleated cells through a constriction, which deforms the cells and causes perturbation of the cells such that antigens and / or tolerogenic factors enter the cells. In some embodiments, the anucleated cells are delivered to an individual and the antigen is delivered to a tolerogenic environment and processed in the tolerogenic environment to induce tolerance to the antigen and / or suppress an immune response to the antigen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 331,368, filed May 3, 2016, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates generally to methods for suppressing immune responses or inducing tolerance by delivering compounds into cells by passing a cell suspension through a cell-deforming constriction. [Background technology]

[0003] The presence of antigens in the non-inflammatory, apoptotic environment of the spleen has been shown to induce anergy and tolerance. One way to ensure consistent presentation of an antigen of interest in this tolerogenic environment is via physical interaction with red blood cells (RBCs).

[0004] However, engineering red blood cells to associate with antigenic material is challenging given that red blood cells are irregularly shaped (biconcave), anucleated, and transcriptionally inactive. As a result, standard transfection techniques have not worked, and therefore proof of the tolerogenic potential of red blood cells initially focused on conjugating materials to the surface of red blood cells (Lorentz et al., Sci. Adv. 2015, 1:e1500112; Grimm et al., Sci Rep. 2015 Oct. 29, 5:15907; Kontos et al., Proc Natl Acad Sci USA. 2013 Jan. 2, 110(1):E60-8). Initial studies using surface conjugation have shown promising results with model antigens and mouse models of type 1 diabetes, but have some notable drawbacks. These drawbacks include: a) the need for chemically modified antigens for attachment, b) limited loading surface area, and c) immunogenicity. Therefore, there is an unmet need for intracellular delivery techniques that can deliver antigens into the cytoplasm of red blood cells and promote a strong immunosuppressive response for the treatment of pathogenic immune responses underlying autoimmune diseases and graft rejection. References that describe methods of using microfluidic constrictions to deliver compounds to cells include WO2013059343, WO2015023982, WO2016070136, WO2016077761, and PCT / US2016 / 13113. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2013 / 059343 [Patent Document 2] International Publication No. 2015 / 023982 [Patent Document 3] International Publication No. 2016 / 070136 [Patent Document 4] International Publication No. 2016 / 077761 [Patent Document 5] International Publication No. 2016 / 115179 [Non-patent literature]

[0006] [Non-Patent Document 1] Lorentz et al., Sci. Adv. 2015, vol. 1: e1500112 [Non-Patent Document 2] Grimm et al., Sci Rep. 2015 October 29, vol. 5:15907 [Non-Patent Document 3] Kontos et al., Proc Natl Acad Sci USA. January 2, 2013, vol. 110(1):E60-8 Summary of the Invention [Means for solving the problem]

[0007] BRIEF SUMMARY OF THE PRESENTINVENTION The present invention provides a method for suppressing an immune response in an individual, comprising the steps of passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that an antigen enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein the antigen is processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0008] Certain aspects of the present invention provide a method for suppressing an immune response in an individual, comprising the steps of passing a cell suspension containing anucleated cells through a constriction, which deforms the cells such that one or more tolerogenic factors enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein the action of the one or more tolerogenic factors contributes to a tolerogenic environment and suppression of the immune response.

[0009] Certain aspects of the present invention provide a method for suppressing an immune response in an individual, comprising the steps of passing a cell suspension containing anucleated cells through a constriction, which deforms the cells such that an antigen and one or more tolerogenic factors enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein processing of the antigen in a tolerogenic environment suppresses the immune response.

[0010] Certain aspects of the present invention provide a method for suppressing an immune response in an individual, comprising the steps of passing a first cell suspension comprising first anucleated cells through a constriction, the constriction deforming the cells such that an antigen enters the anucleated cells, thereby causing perturbation of the cells; passing a second cell suspension comprising second anucleated cells through a constriction, the constriction deforming the cells such that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells; and introducing the first and second anucleated cells into the individual, wherein the antigen is processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0011] Certain aspects of the invention provide a method for suppressing an immune response in an individual comprising the step of introducing into the individual anucleated cell, wherein the anucleated cell comprises an antigen, the antigen being introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, the antigen being processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0012] Certain aspects of the present invention provide a method for delivering a tolerogenic factor to anucleated cells, comprising passing a cell suspension containing the anucleated cells through a constriction, the constriction deforming the anucleated cells such that the tolerogenic factor enters the cells, thereby causing perturbation of the cells, where the cell suspension comes into contact with the tolerogenic factor.

[0013] Certain aspects of the invention provide a method for introducing an antigen into a tolerogenic environment comprising the step of delivering to an individual anucleated cell, wherein the anucleated cell comprises an antigen, and wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, and wherein the antigen is processed in the tolerogenic environment.

[0014] Certain aspects of the invention provide a method for inducing tolerance to an antigen in an individual, comprising the steps of passing a cell suspension comprising anucleated cells through a constriction, which deforms the cells such that antigen enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein the antigen is processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0015] Certain aspects of the invention provide a method for inducing tolerance in an individual, comprising the steps of passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, the action of the tolerogenic factor contributing to a tolerogenic environment that induces tolerance.

[0016] Certain aspects of the invention provide a method for inducing tolerance to an antigen in an individual, comprising the steps of passing a first cell suspension comprising first anucleated cells through a constriction, the constriction deforming the cells so that an antigen enters the anucleated cells, thereby causing perturbation of the cells; passing a second cell suspension comprising second anucleated cells through a constriction, the constriction deforming the cells so that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells; and introducing the first and second anucleated cells into the individual, wherein the antigen is processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0017] Certain aspects of the invention provide a method for inducing tolerance to an antigen in an individual comprising the step of introducing into the individual anucleated cell, wherein the anucleated cell comprises the antigen, the antigen being introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, the antigen being processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0018] Certain aspects of the invention provide a method for delivering an antigen to an anucleated cell, comprising passing a cell suspension comprising the anucleated cells through a constriction, the constriction deforming the anucleated cells such that the antigen enters the cells, thereby causing perturbation of the cells, and the cell suspension contacting the antigen. In some embodiments, the processing and presentation of the antigen in a tolerogenic environment suppresses an immune response to the antigen. In some embodiments, the processing and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments that may be combined with the previous embodiments, the tolerogenic environment is located in the spleen, liver, or lymph nodes.

[0019] In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the channel comprises a constriction width of about 0.25 μm to about 4 μm. In some embodiments, the channel comprises a constriction width of about 4 μm, about 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1 μm, about 0.5 μm, or about 0.25 μm. In some embodiments, the constriction is a pore or is contained within a pore. In some embodiments, the pore is contained in a surface. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the pore size is about 0.5 μm to about 4 μm. In some embodiments, the pore size is 4 μm, about 3 μm, about 2 μm, about 1 μm, or about 0.5 μm. In some embodiments, the constriction size is a function of the diameter of the anucleated cells. In some embodiments, the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70% of the cell diameter. In some embodiments, the method is carried out at between about -5°C and about 45°C.

[0020] References to the diameter of anucleated cells, unless otherwise specified, mean the diameter of the cell in the fluid prior to passing through the constriction, for example as the cell approaches the constriction.

[0021] In some embodiments that may be combined with the previous embodiments, the cell suspension comprises a mixed cell population. In some embodiments, the cell suspension is whole blood. In some embodiments, the cell suspension comprises a purified cell population. In some embodiments, the cell suspension comprises a purified non-nucleated cell population. In some embodiments, the cell suspension comprises mammalian cells. In some embodiments, the cell suspension comprises monkey cells, mouse cells, dog cells, cat cells, horse cells, rat cells, sheep cells, goat cells, pig cells, or rabbit cells. In some embodiments, the cell suspension comprises human cells. In some embodiments, the non-nucleated cells are red blood cells. In some embodiments, the red blood cells are erythrocytes. In some embodiments, the red blood cells are reticulocytes. In some embodiments, the non-nucleated cells are platelets.

[0022] In some embodiments that may be combined with the previous embodiments, the antigen is in a cell lysate. In some embodiments, the antigen is a foreign antigen. In some embodiments, the antigen is an autoantigen. In some embodiments, the antigen is an allogeneic transplant antigen. In some embodiments, the antigen is a microorganism. In some embodiments, the antigen is a lipid antigen. In some embodiments, the antigen is a carbohydrate antigen (e.g., a sugar). In some embodiments, the antigen is a modified antigen. In some embodiments, the modified antigen comprises an antigen fused to a polypeptide. In some embodiments, the modified antigen comprises an antigen fused to a therapeutic agent. In some embodiments, the modified antigen comprises an antigen fused to a targeting peptide. In some embodiments, the modified antigen comprises an antigen fused to a lipid. In some embodiments, the modified antigen comprises an antigen fused to a carbohydrate (e.g., a sugar). In some embodiments, the antigen is an antigen associated with transplant tissue. In some embodiments, the antigen is associated with a virus. In some embodiments, the cell suspension is contacted with the antigen prior to, simultaneously with, or after passage through the constriction.

[0023] In some embodiments that may be combined with the preceding embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα, or TGFβ. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, the polypeptide is a therapeutic peptide. In some embodiments, the polypeptide is conjugated to a carbohydrate (e.g., a sugar). In some embodiments, the cell suspension is contacted with the tolerogenic factor prior to, simultaneously with, or after passing through the constriction. In some embodiments, the half-life of the anucleated cells is decreased. In some embodiments, the half-life of the anucleated cells is increased.

[0024] In embodiments where an immune response is suppressed, the immune response is suppressed by at least about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or more than about 200%. In some embodiments, the suppressed immune response comprises a decrease in T cell response. In some embodiments, the decrease in T cell response comprises a decrease in T cell activation. In some embodiments, the decrease in T cell response comprises a decrease in T cell survival. In some embodiments, the decrease in T cell response comprises a decrease in T cell proliferation. In some embodiments, the decrease in T cell response comprises a decrease in T cell functionality. In some embodiments, the decrease in T cell response comprises a change in T cell phenotype. In some embodiments, the suppressed immune response comprises non-costimulatory activation of T cells. In some embodiments, the suppressed immune response comprises an enhancement of a Treg response. In some embodiments, the suppressed immune response comprises a decrease in a B cell response. In some embodiments, the decrease in a B cell response comprises a decrease in antibody production. In some embodiments, the suppressed immune response comprises a decrease in cytokine production. In some embodiments, the suppressed immune response comprises a reduced autoimmune response. In some embodiments, the suppressed immune response comprises a reduced allergic response. In some embodiments, the suppressed immune response comprises a reduced immune response to the transplanted tissue. In some embodiments, the suppressed immune response comprises a reduced pathogenic immune response to the virus. In some embodiments, the suppressed immune response comprises a reduced immune response to a therapeutic agent. In some embodiments, the suppressed immune response comprises a reduced immune response to a therapeutic vehicle.

[0025] In embodiments where tolerance is induced, the tolerance can include a decrease in T cell response. In some embodiments, the decrease in T cell response can include a decrease in T cell activation. In some embodiments, the decrease in T cell response can include a decrease in T cell survival. In some embodiments, the decrease in T cell response can include a decrease in T cell proliferation. In some embodiments, the decrease in T cell response can include a decrease in T cell functionality. In some embodiments, the decrease in T cell response can include a change in T cell phenotype. In some embodiments, the tolerance can include non-costimulatory activation of T cells. In some embodiments, the tolerance can include an enhanced Treg response. In some embodiments, the tolerance can include a decrease in B cell response. In some embodiments, the decrease in B cell response can include a decrease in antibody production. In some embodiments, the tolerance can include a decrease in cytokine production. In some embodiments, the tolerance can include a decrease in an autoimmune response. In some embodiments, the tolerance can include a decrease in an allergic response. In some embodiments, the tolerance can include a decrease in an immune response to a transplanted tissue. In some embodiments, the tolerance can include a decrease in a pathogenic immune response to the virus. In some embodiments, the tolerance comprises a decreased immune response to the therapeutic agent, hi some embodiments, the tolerance comprises a decreased immune response to the therapeutic vehicle.

[0026] In some embodiments in which modified non-nuclear cells are administered to an individual, the method further comprises at least one (e.g., at least 2, 3, 4, 5, 6 or more) additional administrations of modified non-nuclear cells as described above. In some embodiments, the duration of time between any two administrations is at least 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year.

[0027] Certain aspects of the present invention relate to a system comprising said constriction, a cell suspension and an antigen for use in any one of the methods listed above. Certain aspects of the present invention relate to a system comprising said constriction, a cell suspension and a tolerogenic factor for use in any one of the methods listed above. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. (Item 2) 1. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that tolerogenic factors enter the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the tolerogenic factor contributes to a tolerogenic environment that suppresses the immune response. (Item 3) 1. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigens and tolerogenic factors enter the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. (Item 4) 1. A method for suppressing an immune response in an individual, comprising: a. passing a first cell suspension containing first non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the non-nucleated cells; b. passing a second cell suspension containing second non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the non-nucleated cells; c. introducing the first non-nucleated cell and the second non-nucleated cell into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. (Item 5) 1. A method for suppressing an immune response in an individual comprising the step of introducing into the individual a non-nucleated cell, wherein the non-nucleated cell comprises an antigen, the antigen being introduced into the non-nucleated cell by passing the non-nucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the non-nucleated cell, thereby causing perturbation of the cell, the antigen being processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. (Item 6) 1. A method for suppressing an immune response in an individual, comprising the step of introducing into the individual a non-nucleated cell, wherein the non-nucleated cell comprises an antigen and an immune tolerogenic factor, wherein the antigen and the tolerogenic factor are introduced into the non-nucleated cell by passing the non-nucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the non-nucleated cell, thereby causing perturbation of the cell, wherein the antigen is processed in a tolerogenic environment, and wherein presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. (Item 7) 1. A method for delivering a tolerogenic factor to an anucleated cell, comprising passing a cell suspension containing the anucleated cells through a constriction, the constriction deforming the anucleated cells so that the tolerogenic factor enters the cells, thereby causing perturbation of the cells, wherein the cell suspension comes into contact with the tolerogenic factor. (Item 8) 1. A method for introducing an antigen into a tolerogenic environment, comprising the step of delivering to an individual non-nucleated cells, wherein the non-nucleated cells contain an antigen, and wherein the antigen is introduced into the non-nucleated cells by passing the non-nucleated cells through a constriction, wherein the constriction deforms the cells such that the antigen enters the non-nucleated cells, thereby causing perturbation of the cells, and wherein the antigen is processed in the tolerogenic environment. (Item 9) A method for introducing an antigen and a tolerogenic factor into a tolerogenic environment, comprising the step of delivering to an individual anucleated cell, wherein the anucleated cell comprises the antigen and the tolerogenic factor, and wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing perturbation of the cell, and wherein the antigen is processed in the tolerogenic environment. (Item 10) 1. A method for inducing antigen-specific tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. (Item 11) 1. A method for inducing tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the tolerogenic factor contributes to a tolerogenic environment that induces tolerance. (Item 12) 1. A method for inducing tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigens and tolerogenic factors enter the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. (Item 13) 1. A method for inducing antigen-specific tolerance in an individual, comprising: a. passing a first cell suspension containing first non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the non-nucleated cells; b. passing a second cell suspension containing second non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the non-nucleated cells; c. introducing the first non-nucleated cell and the second non-nucleated cell into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. (Item 14) 1. A method for inducing antigen-specific tolerance in an individual, comprising the step of introducing into the individual anucleated cell, wherein the anucleated cell comprises an antigen, the antigen being introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, the antigen being processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. (Item 15) 1. A method for inducing antigen-specific tolerance in an individual, comprising the step of introducing into the individual anucleated cell, wherein the anucleated cell comprises an antigen and an immune tolerogenic factor, the antigen and the tolerogenic factor being introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing perturbation of the cell, the antigen being processed in a tolerogenic environment, and presentation of the antigen in the tolerogenic environment inducing tolerance to the antigen. (Item 16) 1. A method for delivering an antigen to an anucleated cell, comprising passing a cell suspension containing the anucleated cells through a constriction, the constriction deforming the anucleated cells and thereby causing perturbation of the cells such that the antigen enters the cells, wherein the cell suspension comes into contact with the antigen. (Item 17) 1. A method for delivering an antigen and a tolerogenic factor to an anucleated cell, comprising passing a cell suspension containing the anucleated cells through a constriction, the constriction deforming the anucleated cells and thereby causing perturbation of the cells such that the antigen and the tolerogenic factor enter the cells, wherein the cell suspension comes into contact with the antigen and the tolerogenic factor. (Item 18) 18. The method of claim 16 or 17, wherein processing and presentation of the antigen in a tolerogenic environment suppresses the immune response to the antigen. (Item 19) 18. The method of claim 16 or 17, wherein processing and presentation of the antigen in a tolerogenic environment induces tolerance to the antigen. (Item 20) 20. The method according to any one of items 1, 4, 5, 8, 10 and 13-19, wherein at least one further antigen is introduced into the cells. (Item 21) 18. The method according to any one of items 2 to 4, 6, 7, 9, 11, 12, 13, 15 and 17, wherein at least one further tolerogenic factor is introduced into the cells. (Item 22) 20. The method according to any one of items 1 to 6, 8 to 15, 18 and 19, wherein the tolerogenic environment is located in the spleen, liver or lymph node. (Item 23) 23. The method according to any one of items 1 to 22, wherein the constriction is contained within a microfluidic channel. (Item 24) 24. The method according to any one of items 1 to 23, wherein the channel comprises a constriction width of about 0.25 μm to about 4 μm. (Item 25) 25. The method of any one of items 1 to 24, wherein the channel comprises a constriction width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm or about 0.25 μm. (Item 26) 23. The method according to any one of items 1 to 22, wherein the constriction is a pore or is contained within a pore. (Item 27) 27. The method of claim 26, wherein the pores are contained in a surface. (Item 28) 28. The method of claim 27, wherein the surface is a filter. (Item 29) 28. The method of claim 27, wherein the surface is a membrane. (Item 30) 30. The method according to any one of items 26 to 29, wherein the pore size is from about 0.5 μm to about 4 μm. (Item 31) 31. The method according to any one of items 26 to 30, wherein the pore size is 4 μm, about 3 μm, about 2 μm, about 1 μm or about 0.5 μm. (Item 32) 32. The method according to any one of items 1 to 31, wherein the constriction size is a function of the diameter of the anucleated cells. (Item 33) 33. The method according to any one of items 1 to 32, wherein the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70% of the cell diameter. (Item 34) 34. The method according to any one of items 1 to 33, wherein the method is carried out at a temperature between about -5°C and about 45°C. (Item 35) 35. The method of any one of items 1 to 4, 7, 10 to 13 and 16 to 34, wherein the cell suspension comprises a mixed cell population. (Item 36) 36. The method of claim 35, wherein the cell suspension is whole blood. (Item 37) 35. The method of any one of items 1 to 4, 7, 10 to 13 and 16 to 34, wherein the cell suspension comprises a purified cell population. (Item 38) 38. The method of claim 37, wherein the cell suspension comprises a purified non-nucleated cell population. (Item 39) 39. The method according to any one of items 1 to 4, 7, 10 to 13 and 16 to 38, wherein the cell suspension comprises mammalian cells. (Item 40) 40. The method according to any one of items 1 to 4, 7, 10 to 13 and 16 to 39, wherein the cell suspension comprises monkey cells, mouse cells, dog cells, cat cells, horse cells, rat cells, sheep cells, goat cells, pig cells or rabbit cells. (Item 41) 40. The method according to any one of items 1 to 4, 7, 10 to 13 and 16 to 39, wherein the cell suspension comprises human cells. (Item 42) 42. The method according to any one of items 1 to 41, wherein the anucleated cells are red blood cells. (Item 43) 43. The method of claim 42, wherein the red blood cell is an erythrocyte. (Item 44) 43. The method of claim 42, wherein the red blood cells are reticulocytes. (Item 45) 42. The method according to any one of items 1 to 41, wherein the anucleated cells are platelets. (Item 46) 46. ​​The method according to any one of items 1 to 45, wherein the anuclear cell is a mammalian cell. (Item 47) 47. The method according to any one of items 1 to 46, wherein the anuclear cell is a monkey cell, a mouse cell, a dog cell, a cat cell, a horse cell, a rat cell, a sheep cell, a goat cell, a pig cell or a rabbit cell. (Item 48) 47. The method according to any one of items 1 to 46, wherein the anucleated cells are human cells. (Item 49) The method according to any one of items 1 to 5, 8 to 14 and 22 to 48, wherein the anucleated cells are derived from the individual. (Item 50) 50. The method according to any one of items 1 to 5, 8 to 14 and 22 to 48, wherein the anucleated cells are derived from different individuals. (Item 51) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is in a cell lysate. (Item 52) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a foreign antigen. (Item 53) The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is an autoantigen. (Item 54) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is an allograft antigen. (Item 55) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a protein or a polypeptide. (Item 56) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a lysate. (Item 57) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a microorganism. (Item 58) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a lipid antigen. (Item 59) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is a carbohydrate antigen. (Item 60) 60. The method according to any one of items 1, 4, 5, 8 to 10 and 13 to 59, wherein the antigen is a modified antigen. (Item 61) 61. The method of claim 60, wherein the modified antigen comprises an antigen fused to a polypeptide. (Item 62) 62. The method of item 60 or 61, wherein the modified antigen comprises an antigen fused to a therapeutic agent. (Item 63) 62. The method of claim 60 or 61, wherein the modified antigen comprises an antigen fused to a targeting peptide. (Item 64) 62. The method of claim 60 or 61, wherein the modified antigen comprises an antigen fused to a lipid. (Item 65) 62. The method of claim 60 or 61, wherein the modified antigen comprises an antigen fused to a carbohydrate. (Item 66) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is an antigen associated with transplant tissue. (Item 67) 51. The method according to any one of items 1, 4, 5, 8-10 and 13-50, wherein the antigen is associated with a virus. (Item 68) 68. The method of any one of items 1, 4, 10, 13 and 16-67, wherein the cell suspension is contacted with the antigen prior to, simultaneously with, or after passing through the constriction. (Item 69) 9. The method of any one of items 2 to 4, 7, 11 to 13 and 22 to 68, wherein the tolerogenic factor comprises a polypeptide. (Item 70) 70. The method of claim 69, wherein the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα or TGFβ. (Item 71) 70. The method of claim 69, wherein the polypeptide is a therapeutic polypeptide. (Item 72) 70. The method of claim 69, wherein the polypeptide is a fragment of a therapeutic polypeptide. (Item 73) 70. The method of claim 69, wherein the polypeptide is a therapeutic peptide. (Item 74) 70. The method of claim 69, wherein the polypeptide is conjugated to a carbohydrate. (Item 75) 75. The method according to any one of items 2 to 4, 7, 11 to 13 and 22 to 74, wherein the cell suspension is contacted with the tolerogenic factor prior to, simultaneously with or after passing through the constriction. (Item 76) 76. The method according to any one of items 1 to 75, wherein the half-life of the anucleated cells is decreased. (Item 77) 76. The method according to any one of items 1 to 75, wherein the half-life of the anucleated cells is increased. (Item 78) 78. The method of any one of items 1-6, 18 and 22-77, wherein the immune response is suppressed by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90% or about 100%. (Item 79) 90. The method of any one of items 1 to 6, 18 and 22 to 78, wherein the suppressed immune response comprises reduced production and / or secretion of one or more inflammatory cytokines. (Item 80) 80. The method of claim 79, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). (Item 81) 90. The method of any one of items 1 to 6, 18 and 22 to 78, wherein the suppressed immune response comprises increased production and / or secretion of one or more anti-inflammatory cytokines. (Item 82) 82. The method of claim 81, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor beta (TGFβ). (Item 83) 90. The method of any one of items 1 to 6, 18 and 22 to 78, wherein the suppressed immune response comprises a decrease in T cell response. (Item 84) 84. The method of claim 83, wherein the reduced T cell response comprises reduced T cell activation. (Item 85) 85. The method of item 83 or 84, wherein the reduced T cell response comprises reduced T cell survival. (Item 86) 86. The method of any one of items 83 to 85, wherein the reduced T cell response comprises reduced T cell proliferation. (Item 87) 87. The method of any one of items 83 to 86, wherein the reduced T cell response comprises reduced T cell functionality. (Item 88) 88. The method of any one of items 83 to 87, wherein the reduction in T cell response comprises a change in T cell phenotype. (Item 89) 90. The method of any one of items 1 to 6, 18 and 22 to 88, wherein the immune response that is suppressed comprises non-costimulatory activation of T cells. (Item 90) 90. The method of any one of items 1 to 6, 18 and 22 to 89, wherein the suppressed immune response comprises an enhancement of a Treg response. (Item 91) The method of any one of items 1 to 6, 18 and 22 to 90, wherein the suppressed immune response comprises a decrease in a B cell response. (Item 92) 92. The method of claim 91, wherein the reduced B cell response comprises reduced antibody production. (Item 93) 93. The method of any one of items 1 to 6, 18 and 22 to 92, wherein the suppressed immune response comprises a decrease in cytokine production. (Item 94) 94. The method of any one of items 1 to 6, 18 and 22 to 93, wherein the suppressed immune response comprises a reduction in an autoimmune response. (Item 95) 5. The method of any one of items 1 to 6, 18 and 22 to 94, wherein the suppressed immune response comprises a reduction in an allergic response. (Item 96) The method of any one of items 1 to 6, 18 and 22 to 95, wherein the suppressed immune response comprises a decrease in the immune response to the transplanted tissue. (Item 97) 96. The method of any one of items 1 to 6, 18 and 22 to 95, wherein the suppressed immune response comprises a reduction in a pathogenic immune response to the virus. (Item 98) 96. The method of any one of items 1-6, 18 and 22-95, wherein the suppressed immune response comprises a decrease in the immune response to a therapeutic agent. (Item 99) The method of any one of items 1 to 6, 18 and 22 to 95, wherein the suppressed immune response comprises a decrease in the immune response to a therapeutic vehicle. (Item 100) 8. The method according to any one of items 10 to 15 and 19 to 77, wherein said tolerance comprises reduced production and / or secretion of one or more inflammatory cytokines. (Item 101) 101. The method of claim 100, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF). (Item 102) 78. The method of any one of items 10 to 15 and 19 to 77, wherein said tolerance comprises increased production and / or secretion of one or more anti-inflammatory cytokines. (Item 103) 103. The method of claim 102, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor beta (TGFβ). (Item 104) 78. The method of any one of items 10 to 15 and 19 to 77, wherein the tolerance comprises a decrease in T cell responses. (Item 105) 105. The method of claim 104, wherein the reduced T cell response comprises reduced T cell activation. (Item 106) 106. The method of claim 104 or 105, wherein the reduced T cell response comprises reduced T cell survival. (Item 107) 107. The method of any one of items 104 to 106, wherein the reduction in T cell response comprises a reduction in T cell proliferation. (Item 108) 108. The method according to any one of items 104 to 107, wherein the reduction in T cell response comprises a reduction in T cell functionality. (Item 109) 109. The method of any one of items 104 to 108, wherein the reduction in T cell response comprises a change in T cell phenotype. (Item 110) The method of any one of items 10 to 15, 19 to 77 and 104 to 109, wherein the tolerance comprises non-costimulatory activation of T cells. (Item 111) The method of any one of items 10 to 15, 19 to 77 and 104 to 110, wherein the tolerance comprises an enhancement of a Treg response. (Item 112) The method of any one of items 10 to 15, 19 to 77 and 104 to 111, wherein the tolerance comprises a decrease in B cell responses. (Item 113) 13. The method of claim 112, wherein the reduced B cell response comprises reduced antibody production. (Item 114) The method of any one of items 10 to 15, 19 to 77 and 104 to 113, wherein the tolerance comprises a decrease in cytokine production. (Item 115) The method of any one of items 10 to 15, 19 to 77 and 104 to 114, wherein the tolerance comprises a reduction in an autoimmune response. (Item 116) 116. The method of any one of items 10 to 15, 19 to 77 and 104 to 115, wherein said tolerance comprises a reduction in an allergic response. (Item 117) The method of any one of items 10 to 15, 19 to 77 and 104 to 116, wherein the tolerance comprises a decrease in the immune response to the transplanted tissue. (Item 118) 117. The method of any one of items 10 to 15, 19 to 77 and 104 to 116, wherein the tolerance comprises a reduction in a pathogenic immune response to the virus. (Item 119) The method of any one of items 10 to 15, 19 to 77 and 104 to 116, wherein the tolerance comprises a decreased immune response to the therapeutic agent. (Item 120) The method of any one of items 10 to 15, 19 to 77 and 104 to 116, wherein the tolerance comprises a decrease in the immune response to the therapeutic vehicle. (Item 121) 121. The method of any one of items 1 to 120, repeated at least 1, 2, 3, 4, 5 or 6 times. (Item 122) 122. The method according to item 121, wherein the time interval between any two repetitions of said method is at least one day, one week, one month, two months, three months, four months, five months, six months or one year. (Item 123) 121. A system comprising the constriction, a cell suspension and an antigen for use in the method according to any one of items 1, 4, 10, 13 and 16 to 120. (Item 124) A system comprising the constriction, a cell suspension and a tolerogenic factor for use in the method according to any one of items 2 to 4, 7, 11 to 13 and 22 to 120. [Brief description of the drawings]

[0028] [Figure 1] 1A and 1B show exemplary flow cytometry histogram plots showing fluorescence following constriction-mediated delivery (SQZ) of IgG antibodies (FIG. 1A) and dextran particles (FIG. 1B) to human RBCs compared to endocytosis controls.

[0029] [Diagram 2] Figure 2A shows the delivery efficiency after constriction-mediated delivery of IgG antibody and dextran particles to human RBCs, and Figure 2B shows the estimated cell viability after constriction-mediated delivery of IgG antibody and dextran particles to human RBCs.

[0030] [Figure 3A] Figure 3A shows an exemplary flow cytometry histogram plot showing fluorescence after constriction-mediated delivery of an IgG antibody to mouse RBCs. Figure 3B shows estimated cell viability after constriction-mediated delivery of an IgG antibody to mouse RBCs. Figure 3C shows delivery efficiency after constriction-mediated delivery of an IgG antibody to mouse RBCs. [Figure 3BC] Figure 3A shows an exemplary flow cytometry histogram plot showing fluorescence after constriction-mediated delivery of an IgG antibody to mouse RBCs. Figure 3B shows estimated cell viability after constriction-mediated delivery of an IgG antibody to mouse RBCs. Figure 3C shows delivery efficiency after constriction-mediated delivery of an IgG antibody to mouse RBCs.

[0031] [Figure 4ABC]Figures 4A-I show exemplary FACS plots demonstrating delivery of dextran particles to mouse RBCs at 2 psi (Figure 4D), 4 psi (Figure 4E), 6 psi (Figure 4F), 10 psi (Figure 4G), 20 psi (Figure 4H), or using manual syringe pressure (Figure 4I) compared to endocytosis (Figure 4A), negative control (Figure 4B), and no material control (Figure 4C). [Figure 4DEF] Figures 4A-I show exemplary FACS plots demonstrating delivery of dextran particles to mouse RBCs at 2 psi (Figure 4D), 4 psi (Figure 4E), 6 psi (Figure 4F), 10 psi (Figure 4G), 20 psi (Figure 4H), or using manual syringe pressure (Figure 4I) compared to endocytosis (Figure 4A), negative control (Figure 4B), and no material control (Figure 4C). [Figure 4GHI] Figures 4A-I show exemplary FACS plots demonstrating delivery of dextran particles to mouse RBCs at 2 psi (Figure 4D), 4 psi (Figure 4E), 6 psi (Figure 4F), 10 psi (Figure 4G), 20 psi (Figure 4H), or using manual syringe pressure (Figure 4I) compared to endocytosis (Figure 4A), negative control (Figure 4B), and no material control (Figure 4C).

[0032] [Figure 5A] Figure 5A shows the estimated cell viability after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 5B shows the delivery efficiency after stenosis-mediated delivery of IgG antibodies or dextran particles to mouse RBCs. Figure 5C shows the geometric mean fluorescence after stenosis-mediated delivery of IgG antibodies or dextran particles to mouse RBCs. [Figure 5BC] Figure 5A shows the estimated cell viability after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 5B shows the delivery efficiency after stenosis-mediated delivery of IgG antibodies or dextran particles to mouse RBCs. Figure 5C shows the geometric mean fluorescence after stenosis-mediated delivery of IgG antibodies or dextran particles to mouse RBCs.

[0033] [Figure 6AB]Figure 6A shows an exemplary flow cytometry histogram plot showing fluorescence after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6B shows estimated cell viability after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6C shows delivery efficiency after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6D shows the geometric mean fluorescence after stenosis-mediated delivery of dextran particles to mouse RBCs. [Figure 6C-D] Figure 6A shows an exemplary flow cytometry histogram plot showing fluorescence after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6B shows estimated cell viability after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6C shows delivery efficiency after stenosis-mediated delivery of dextran particles to mouse RBCs. Figure 6D shows the geometric mean fluorescence after stenosis-mediated delivery of dextran particles to mouse RBCs.

[0034] [Figure 7] FIG. 7 shows a diagram of the treatment schedule.

[0035] [Figure 8] FIG. 8 shows the numbers of OT-I-specific T cells in mice from the four treatment groups.

[0036] [Figure 9] FIG. 9 shows the percentage of splenic T cells that expressed high levels of IFN-γ (left) and the levels of IFN-γ production (right).

[0037] [Figure 10] FIG. 10 shows representative flow cytograms of the percentage of OT-I T cells among all CD8+ T cells (upper panel) and the percentage of IFN-γ-responsive T cells among OT-I T cells (lower panel).

[0038] [Figure 11] 11A and 11B show diagrams of a representative treatment schedule.

[0039] [Figure 12] 12A and 12B show diagrams of representative treatment schedules. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present invention provides methods of inducing tolerance and / or suppressing an immune response in an individual by passing a cell suspension containing anucleated cells through a constriction to allow delivery of antigens and / or tolerogenic factors to the anucleated cells. In some embodiments, the constriction is contained within a microfluidic channel. In some embodiments, the constriction is a pore or is contained within a pore.

[0041] Certain aspects of the present disclosure relate to a method for inducing tolerance to an antigen in an individual and / or suppressing an immune response to said antigen, comprising passing a cell suspension containing anucleated cells through a constriction, which deforms the cells such that the antigen enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein presentation of the antigen in a tolerogenic environment induces tolerance to the antigen and / or suppresses an immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the tolerance and / or immune suppression is antigen-specific. In some embodiments, the tolerance and / or immune suppression is non-specific and includes tolerance to and / or suppression of immune responses to multiple antigens.

[0042] Certain aspects of the present disclosure relate to a method for inducing tolerance to an antigen and / or suppressing an immune response to an antigen in an individual, comprising passing a cell suspension containing anucleated cells through a constriction, which deforms the cells such that antigens and tolerogenic factors contacted with the cells enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby inducing tolerance to the antigen and / or suppressing an immune response to the antigen. In some embodiments, the antigen is presented in a tolerogenic environment. In some embodiments, the tolerogenic factors generate or promote a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen and / or suppresses an immune response to the antigen. For example, in some embodiments, the tolerogenic factors act in the tolerogenic environment to further increase the tolerogenic nature of the environment. In some embodiments, the tolerogenic factor acts in a non-tolerogenic environment to generate a tolerogenic environment. In some embodiments, the antigen is processed in a tolerogenic environment. In some embodiments, the tolerance and / or immune suppression is antigen-specific. In some embodiments, the tolerance and / or immune suppression is non-specific and includes tolerance to multiple antigens and / or suppression of immune responses.

[0043] Certain aspects of the present disclosure relate to a method for inducing tolerance to an antigen and / or suppressing an immune response to an antigen in an individual, comprising passing a cell suspension containing anucleated cells through a constriction, which deforms the cells such that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby inducing tolerance to the antigen and / or suppressing the immune response to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of an antigen in the tolerogenic environment induces tolerance to the antigen and / or suppresses the immune response to the antigen. In some embodiments, the tolerance and / or immune suppression is antigen-specific. In some embodiments, the tolerance and / or immune suppression is non-specific and includes tolerance to and / or suppression of immune responses to multiple antigens.

[0044] I. General technology The techniques and procedures described or referenced herein are generally well understood and may be readily understood by those of skill in the art using conventional methodology, e.g., Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (eds. FM Ausubel et al., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (eds. MJ MacPherson, BD Hames and GR Taylor, 1995); Antibodies, A Laboratory Manual (eds. Harlow and Lane, 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (eds. RI Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (ed. MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (ed. J. E. Cellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (ed. J. P. Mather and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (eds. A. Doyle, J. B. Griffiths and D. G. Newell, J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (ed. D. M. Weir and CCBlackwell, 1996); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, 1987); PCR: The Polymerase Chain Reaction (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011).

[0045] II. Definition For purposes of interpretation of this specification, the following definitions shall apply and, where applicable, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0046] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise.

[0047] Aspects and embodiments of the invention described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0048] The term "about" as used herein refers to the normal error range for the respective value, which is readily apparent to one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to the value or parameter itself.

[0049] The term "pore" as used herein refers to an opening, including but not limited to a hole, a crevice, a cavity, an opening, a crack, a gap, or a perforation in a material. In some examples (where indicated), the term refers to a hole in a surface of the present disclosure. In other examples (where indicated), the pore can refer to a hole in a cell membrane.

[0050] The term "membrane" as used herein refers to a selective barrier or sheet containing pores. The term includes flexible sheet-like structures that act as a boundary or lining. In some instances, the term refers to a surface or filter that contains pores. This term is distinct from the term "cell membrane."

[0051] The term "filter" as used herein refers to a porous article that allows selective passage through the pores. In some instances, the term refers to a surface or membrane that contains pores.

[0052] The term "heterogeneous" as used herein refers to something that is mixed or non-uniform in structure or composition. In some instances, the term refers to pores having various sizes, shapes, or distributions within a given surface.

[0053] The term "uniform" as used herein refers to something that is consistent or uniform throughout its structure or composition. In some instances, the term refers to pores that have a consistent size, shape, or distribution within a given surface.

[0054] The term "heterologous" as used herein refers to a molecule derived from a different organism. In some instances, the term refers to a nucleic acid or protein that is not normally found or expressed in a given organism.

[0055] As used herein, the term "inhibit" can refer to the action of blocking, reducing, eliminating, or otherwise antagonizing the presence or activity of a particular target. Inhibition can refer to partial or complete inhibition. For example, inhibiting an immune response can refer to any action that leads to blocking, reducing, eliminating, or any other antagonism of an immune response. In other examples, inhibiting expression of a nucleic acid can include, but is not limited to, reducing transcription of the nucleic acid, reducing mRNA abundance (e.g., mRNA transcription silencing), degradation of mRNA, inhibiting mRNA translation, gene editing, and the like. In other examples, inhibiting expression of a protein can include, but is not limited to, reducing transcription of a nucleic acid encoding a protein, reducing stability of an mRNA encoding a protein, inhibiting translation of a protein, reducing stability of a protein, and the like.

[0056] As used herein, the term "suppress" can refer to the action of decreasing, reducing, preventing, limiting, decreasing, or otherwise attenuating the presence or activity of a particular target. Suppression can refer to partial or complete suppression. For example, suppressing an immune response can refer to any action that leads to decreasing, reducing, preventing, limiting, decreasing, or otherwise attenuating an immune response. In other examples, suppressing the expression of a nucleic acid can include, but is not limited to, reducing the transcription of the nucleic acid, reducing mRNA abundance (e.g., mRNA transcription silencing), degrading the mRNA, inhibiting mRNA translation, and the like. In other examples, suppressing the expression of a protein can include, but is not limited to, reducing the transcription of a nucleic acid encoding a protein, reducing the stability of an mRNA encoding a protein, inhibiting the translation of a protein, reducing the stability of a protein, and the like.

[0057] As used herein, the term "enhancing" can refer to the act of improving, enhancing, strengthening, or otherwise increasing the presence or activity of a particular target. For example, enhancing an immune response can refer to any action that leads to improving, enhancing, strengthening, or otherwise increasing an immune response. In other examples, enhancing expression of a nucleic acid can include, but is not limited to, increasing transcription of the nucleic acid, increasing mRNA abundance (e.g., increasing transcription of the mRNA), decreasing mRNA degradation, increasing mRNA translation, and the like. In other examples, enhancing expression of a protein can include, but is not limited to, increasing transcription of a nucleic acid encoding a protein, increasing stability of an mRNA encoding a protein, increasing translation of a protein, increasing stability of a protein, and the like.

[0058] As used herein, the term "induce" can refer to the action of initiating, promoting, stimulating, establishing, or otherwise causing a result. For example, inducing an immune response can refer to any action that leads to initiating, promoting, stimulating, establishing, or otherwise causing a desired immune response. In other examples, inducing expression of a nucleic acid can include, but is not limited to, initiating transcription of a nucleic acid, initiating translation of an mRNA, and the like. In other examples, inducing expression of a protein can include, but is not limited to, increasing transcription of a nucleic acid encoding a protein, increasing stability of an mRNA encoding a protein, increasing translation of a protein, increasing stability of a protein, and the like.

[0059] The term "homologous" as used herein refers to molecules that are derived from the same organism. In some instances, the term refers to nucleic acids or proteins that are normally found or expressed in a given organism.

[0060] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of a polynucleotide can include sugar and phosphate groups (as may be commonly found in RNA or DNA), or modified or substituted sugar or phosphate groups. The backbone of a polynucleotide can include repeating units such as N-(2-aminoethyl)-glycine linked by peptide bonds (i.e., peptide nucleic acid). Alternatively, the backbone of a polynucleotide can include polymers of synthetic subunits such as phosphoramidates, and thus may be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. Additionally, double-stranded polynucleotides can be obtained from single-stranded polynucleotide products of chemical synthesis by either synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0061] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins that contain modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. These modifications may be deliberate, through site-directed mutagenesis, or may be accidental, for example, through mutations of the host that produces the protein or errors due to PCR amplification.

[0062] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0063] III. Immunosuppression and Tolerance In certain aspects, the present invention provides a method for suppressing an immune response and / or inducing tolerance in an individual, comprising the steps of passing a cell suspension comprising anucleated cells through a constriction, which deforms the cells such that antigens and / or tolerogenic compounds enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein the antigens are processed in a tolerogenic environment, and presentation of the antigens in the tolerogenic environment suppresses an immune response to the antigens.

[0064] Immunological tolerance is immunological unresponsiveness to challenge with an antigen to which tolerance was induced. Tolerance is indicated when a subject is unresponsive to subsequent challenge with the tolerance-inducing antigen. Tolerance can be mediated by several different mechanisms, including apoptosis of autoreactive cells, induction of anergy, or deviation of lymphocyte phenotype. In some embodiments, the tolerance can include T cell unresponsiveness, which causes deactivation of the immune response to a specific antigen. Tolerance can also be mediated by regulatory T cells (Tregs) through secretion of immunosuppressive cytokines and through contact-dependent interactions with cell surface molecules or cytotoxic factors. Defects in tolerogenic mechanisms can contribute to autoimmune diseases, transplant rejection, anti-drug responses, and pathogenic responses to viral infections. Immunosuppression is a decrease in the activity of the immune response. For example, immunosuppression can include, but is not limited to, decreased responsiveness to challenge with an antigen, decreased immune cell activation and proliferation, modulation of cytokine secretion, decreased immune cell survival, or decreased immune cell effector function. In some embodiments, the immune suppression is specific for a given antigen, e.g., an immune response to a self-antigen is suppressed.

[0065] In some embodiments, the antigen is processed and presented in a tolerogenic environment. The tolerogenic environment may be a location in the body that supports the development of a tolerogenic immune response. In some embodiments, the tolerogenic environment is located in a primary lymphoid tissue, such as the thymus. In some embodiments, the tolerogenic environment is located in a secondary lymphoid tissue. Examples of secondary lymphoid tissues include the spleen, lymph nodes, and mucosa-associated lymphoid tissue (MALT). In some embodiments, the tolerogenic environment is located in the spleen. In some embodiments, the tolerogenic environment is located in a non-lymphoid tissue, such as the liver. In some embodiments, the tolerogenic environment is induced at an inflammation site by delivery of a tolerogenic factor. For example, an inflammatory environment is converted to a tolerogenic environment by a tolerogenic factor.

[0066] In some embodiments, the present invention provides a method for delivery to an anucleated cell, the cell being a mammalian cell. An anucleated cell lacks a nucleus. In some embodiments, the anucleated cell is a monkey cell, a mouse cell, a dog cell, a cat cell, a horse cell, a rat cell, a sheep cell, a goat cell, a pig cell, or a rabbit cell. In some embodiments, the anucleated cell is a human cell. In some embodiments, the anucleated cell is a non-mammalian cell. In some embodiments, the anucleated cell is a chicken cell, a frog cell, an insect cell, a fish cell, or a nematode cell. In some embodiments, the anucleated cell is a red blood cell. Red blood cells (RBCs) are flexible, elliptical, biconcave discs with a cytoplasm rich in the oxygen-carrier biomolecule hemoglobin. RBCs serve as the primary vehicle for oxygen delivery and carbon dioxide removal throughout the human body. RBCs can remain in circulation for up to 120 days, after which they are removed from the body by clearance in the spleen. Reticulocytes are anucleated immature (not yet biconcave) red blood cells, typically constituting about 1% of the red blood cells in the human body. Mature red blood cells are also called erythrocytes. In some embodiments, the anucleated cells are platelets. Platelets, also called thrombocytes, are components of blood whose function is related to blood clotting. Platelets are biconvex discoid (lenticular) structures with a diameter of 2-3 μm.

[0067] In some embodiments, presentation of an antigen in the tolerogenic environment suppresses an immune response to the antigen or induces a tolerogenic response to the antigen. Antigens from apoptotic cells, e.g., RBCs, that are normally removed in the tolerogenic environment of the spleen, can drive tolerance to the antigen and / or suppress immune responses to the antigen through deletion (e.g., killing) or anergy of reactive T cells. In some embodiments, the tolerance and / or immune suppression is antigen-specific. RBCs have a limited life span and cannot repair themselves, resulting in RBC death by eryptosis, a process similar to apoptosis, followed by removal from the bloodstream. In some embodiments, the antigen may be released upon apoptosis of the anucleated cells in the tolerogenic environment, which are then taken up, processed, and presented by antigen-presenting cells. In some embodiments, the anucleated cells containing the antigen are phagocytosed by antigen-presenting cells, e.g., macrophages, and the antigen is then processed and presented by the antigen-presenting cells.

[0068] In some embodiments, the half-life of the anucleate can be modified. In some embodiments, the half-life of the anucleate is increased. For example, the anucleate can be modified to increase the time the anucleate circulates in the bloodstream before clearance in the spleen. In some embodiments, the half-life of the anucleate is decreased. For example, the anucleate can be modified to decrease the time the anucleate circulates in the bloodstream before clearance in the spleen. In some embodiments, altering the lipid ratio on the anucleate surface decreases the half-life of the anucleate. For example, the presence of phosphatidylserine on the surface of the anucleate can be increased to decrease the half-life of the anucleate, for example, by using any method known in the art for increasing surface phosphatidylserine (see Hamidi et al., J. Control. Release, 2007, vol. 118(2):145-60). Phosphatidylserine exposure on the outer cell membrane is a hallmark of apoptosis and is recognized by receptors on phagocytes in a manner that facilitates uptake. In some embodiments, the anucleated cells are incubated with lipids prior to delivery to an individual. In some embodiments, the anucleated cells are associated with a polymer to reduce the half-life of the anucleated cells.

[0069] In certain aspects, the invention provides a method for suppressing an immune response to an antigen in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that antigen enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein presentation of the antigen in a tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the immune response is antigen-specific.

[0070] In certain aspects, the invention provides a method for suppressing an immune response in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby suppressing the immune response. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the immune suppression is non-specific and includes suppression of immune responses to multiple antigens.

[0071] In certain aspects, the present invention provides a method for suppressing an immune response to an antigen in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that antigen and tolerogenic factors enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby suppressing the immune response to the antigen. In some embodiments, the antigen is presented in a tolerogenic environment. In some embodiments, the tolerogenic factors generate or promote a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the immune suppression is antigen-specific. In some embodiments, the immune suppression is non-specific and includes suppression of immune responses to multiple antigens.

[0072] In certain aspects, the present invention provides a method for suppressing an immune response to an antigen in an individual, comprising the steps of: passing a first cell suspension comprising first anucleated cells through a constriction, the constriction deforming the cells so that an antigen enters the anucleated cells, thereby causing perturbation of the cells; passing a second cell suspension comprising second anucleated cells through a constriction, the constriction deforming the cells so that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells; and introducing the first anucleated cells and the second anucleated cells into the individual, thereby suppressing an immune response to the antigen. In some embodiments, the antigen is presented in a tolerogenic environment. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the first and second anuclear cells are introduced simultaneously. In some embodiments, the first and second anuclear cells are introduced sequentially. In some embodiments, the first anuclear cells are introduced into the individual before the introduction of the second anuclear cells. In some embodiments, the first anuclear cells are introduced into the individual more than about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the second anuclear cells. In some embodiments, the second anuclear cells are introduced into the individual before the introduction of the first anuclear cells. In some embodiments, the second anuclear cells are introduced into the individual more than about 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the first anuclear cells. In some embodiments, the immune suppression is antigen-specific, hi some embodiments, the immune suppression is non-specific and includes suppression of the immune response to multiple antigens.

[0073] In certain aspects, the invention provides a method for inducing tolerance to an antigen in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that antigen enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, wherein presentation of the antigen in a tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the tolerance is antigen-specific.

[0074] In certain aspects, the invention provides a method for inducing tolerance to an antigen in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the cells such that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby inducing tolerance to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments, the tolerance is non-specific and includes tolerance to multiple antigens.

[0075] In certain aspects, the present invention provides a method for inducing tolerance to an antigen in an individual, comprising passing a cell suspension comprising anucleated cells through a constriction, which deforms the cells such that antigen and tolerogenic factors enter the anucleated cells, thereby causing perturbation of the cells, and introducing the anucleated cells into the individual, thereby inducing tolerance to the antigen. In some embodiments, the antigen is presented in a tolerogenic environment. In some embodiments, the tolerogenic factors generate or promote a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the tolerance is antigen-specific. In some embodiments, the tolerance is non-specific and includes tolerance to multiple antigens.

[0076] In certain aspects, the present invention provides a method for inducing tolerance to an antigen in an individual, comprising the steps of passing a first cell suspension comprising a first anucleated cell through a constriction, the constriction deforming the cells so that an antigen enters the anucleated cells, thereby causing perturbation of the cells, passing a second cell suspension comprising a second anucleated cell through a constriction, the constriction deforming the cells so that a tolerogenic factor enters the anucleated cells, thereby causing perturbation of the cells, and introducing the first and second anucleated cells into the individual, thereby inducing tolerance to the antigen. In some embodiments, the antigen is presented in a tolerogenic environment. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the first and second anuclear cells are introduced simultaneously. In some embodiments, the first and second anuclear cells are introduced sequentially. In some embodiments, the first anuclear cells are introduced into the individual before the introduction of the second anuclear cells. In some embodiments, the first anuclear cells are introduced into the individual more than about any of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the second anuclear cells. In some embodiments, the second anuclear cells are introduced into the individual before the introduction of the first anuclear cells. In some embodiments, the second anuclear cells are introduced into the individual more than about any of 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the introduction of the first anuclear cells. In some embodiments, the tolerance is antigen-specific, hi some embodiments, the tolerance is non-specific and includes tolerance to multiple antigens.

[0077] In certain aspects, the invention provides a method for inducing tolerance to an antigen in an individual, comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises the antigen, and wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, and wherein presentation of the antigen in a tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the tolerance is antigen-specific.

[0078] In certain aspects, the invention provides a method for suppressing an immune response to an antigen in an individual, comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises the antigen, and wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, and wherein presentation of the antigen in a tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the immune suppression is antigen-specific.

[0079] In certain aspects, the invention provides a method for inducing tolerance to an antigen in an individual, comprising introducing into the individual anucleated cell, wherein the anucleated cell comprises a tolerogenic factor, the tolerogenic factor being introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the tolerogenic factor enters the anucleated cell, thereby causing a perturbation of the cell, thereby inducing tolerance to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments, the tolerance is non-specific and includes tolerance to multiple antigens.

[0080] In certain aspects, the invention provides a method for suppressing an immune response to an antigen in an individual, comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises a tolerogenic factor, and wherein the tolerogenic factor is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the tolerogenic factor enters the anucleated cell, thereby causing perturbation of the cell, thereby suppressing the immune response to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the immune suppression is non-specific and includes suppression of immune responses to multiple antigens.

[0081] In certain aspects, the invention provides a method for inducing tolerance to an antigen in an individual, comprising introducing an anucleated cell into the individual, wherein the anucleated cell comprises an antigen and a tolerogenic factor, and wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing a perturbation of the cell, and presentation of the antigen induces tolerance to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the tolerance is antigen-specific. In some embodiments, the tolerance is non-specific and includes tolerance to multiple antigens.

[0082] In certain aspects, the invention provides a method for suppressing an immune response to an antigen in an individual, comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises an antigen and a tolerogenic factor, and wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing a perturbation of the cell, and wherein presentation of the antigen suppresses the immune response to the antigen. In some embodiments, the tolerogenic factor generates or promotes a tolerogenic environment, and presentation of the antigen in the tolerogenic environment suppresses the immune response to the antigen. In some embodiments, the antigen is processed in the tolerogenic environment. In some embodiments, the immune suppression is antigen-specific. In some embodiments, the immune suppression is non-specific and includes suppression of immune responses to multiple antigens.

[0083] In certain aspects, the invention provides methods for introducing an antigen into a tolerogenic environment in an individual comprising delivering to the individual anucleated cell, wherein the anucleated cell comprises the antigen, and wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell. In some embodiments, the antigen is presented in the tolerogenic environment. In some embodiments, the antigen is processed in the tolerogenic environment.

[0084] In certain aspects, the invention provides methods for generating a tolerogenic environment in an individual comprising delivering to the individual an anucleated cell, wherein the anucleated cell comprises a tolerogenic factor, and wherein the tolerogenic factor is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the tolerogenic factor enters the anucleated cell, thereby causing perturbation of the cell. In some embodiments, presentation of the antigen in the tolerogenic environment induces tolerance to the antigen and / or suppresses an immune response to the antigen. In some embodiments, the tolerance and / or immune suppression is non-specific and includes tolerance to and / or suppression of immune responses to multiple antigens.

[0085] In some embodiments, the immune response is suppressed by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90%, or about 100%. In some embodiments, the suppressed immune response and / or induced tolerance comprises a decrease in T cell response. For example, a decrease in T cell response can include, but is not limited to, a decrease in T cell activation or proliferation, a decrease in T cell survival, or a decrease in cell functionality. In some embodiments, the decrease in T cell response comprises a decrease in T cell activation. In some embodiments, the decrease in T cell response comprises a decrease in T cell survival. In some embodiments, the decrease in T cell response comprises a decrease in T cell proliferation. In some embodiments, the decrease in T cell response comprises a decrease in T cell functionality. For example, a decrease in T cell functionality can include, but is not limited to, a modulation of cytokine secretion, a decrease in T cell migration to sites of inflammation, and a decrease in T cell cytotoxic activity. In some embodiments, the suppressed immune response and / or induced tolerance comprises a decrease in the production and / or secretion of inflammatory cytokines and / or an increase in the production and / or secretion of anti-inflammatory cytokines. In some embodiments, the suppressed immune response and / or induced tolerance comprises a decrease in the production and / or secretion of one or more pro-inflammatory cytokines selected from interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the suppressed immune response and / or induced tolerance comprises a decrease in the production and / or secretion of one or more anti-inflammatory cytokines selected from IL-4, IL-10, IL-13, IL-35, IFN-alpha and transforming growth factor beta (TGFβ). In some embodiments, the suppressed immune response and / or induced tolerance comprises a change in T cell phenotype. For example, the state of the T cells can be changed from a pro-inflammatory phenotype to a regulatory or anti-inflammatory phenotype.In some embodiments, the suppressed immune response and / or induced tolerance comprises non-costimulatory activation of T cells, which may subsequently lead to cell death. In some embodiments, the suppressed immune response and / or induced tolerance comprises an enhanced Treg response. In some embodiments, the suppressed immune response and / or induced tolerance comprises a decrease in a B cell response. In some embodiments, the decrease in a B cell response comprises a decrease in antibody production.

[0086] In some embodiments, the immune response suppressed and / or tolerance induced comprises a reduction in an autoimmune response. For example, the reduction in the autoimmune response can include, but is not limited to, a reduction in an immune response or induction of tolerance to an antigen associated with type I diabetes; rheumatoid arthritis; psoriasis; multiple sclerosis; neurodegenerative diseases that may have an immune component, such as Alzheimer's disease, ALS, Huntington's disease, and Parkinson's disease; systemic lupus erythematosus; Sjogren's syndrome; Crohn's disease, or ulcerative colitis. In some embodiments, the immune response suppressed and / or tolerance induced comprises a reduction in an allergic response. For example, the reduction in the allergic response can include a reduction in an immune response or induction of tolerance to an antigen associated with allergic asthma, atopic dermatitis, allergic rhinitis (hay fever), or food allergy. In some embodiments, the antigen is an antigen associated with a transplanted tissue. In some embodiments, the immune response suppressed and / or tolerance induced comprises a reduction in an immune response or induction of tolerance to the transplanted tissue. In some embodiments, the antigen is associated with a virus. In some embodiments, the suppressed immune response and / or induced tolerance comprises a reduction in a pathogenic immune response to the virus or induction of tolerance to the virus. For example, the pathogenic immune response may comprise a cytokine storm generated by certain viral infections. A cytokine storm is a potentially lethal immune response that consists of a positive feedback loop between cytokines and white blood cells.

[0087] In some embodiments, the suppressed immune response comprises a decrease in immune response to a therapeutic agent. In some embodiments, the therapeutic agent is a clotting factor. Exemplary clotting factors include, but are not limited to, Factor VIII and Factor IX. In some embodiments, the therapeutic agent is an antibody. Exemplary therapeutic antibodies include, but are not limited to, anti-TNFα, anti-VEGF, anti-CD3, anti-CD20, anti-IL-2R, anti-Her2, anti-RSVF, anti-CEA, anti-IL-1 beta, anti-CD15, anti-myosin, anti-PSMA, anti-40kDa glycoprotein, anti-CD33, anti-CD52, anti-IgE, anti-CD11a, anti-EGFR, anti-C5, anti-alpha-4 integrin, anti-IL-12 / IL-23, anti-IL-6R, and anti-RANKL. In some embodiments, the therapeutic agent is a growth factor. Exemplary therapeutic growth factors include, but are not limited to, erythropoietin (EPO) and megakaryocyte differentiation and growth factor (MDGF). In some embodiments, the therapeutic agent is a hormone. Exemplary therapeutic hormones include, but are not limited to, insulin, human growth hormone, and follicle stimulating hormone. In some embodiments, the therapeutic agent is a recombinant cytokine. Exemplary therapeutic recombinant cytokines include, but are not limited to, IFNβ, IFNα, and GM-CSF. In some embodiments, the suppressed immune response includes a decrease in immune response to a therapeutic vehicle. In some embodiments, the therapeutic vehicle is a virus used for gene therapy, such as an adenovirus, an adeno-associated virus, a baculovirus, a herpes virus, or a retrovirus. In some embodiments, the therapeutic vehicle is a liposome. In some embodiments, the therapeutic vehicle is a nanoparticle.

[0088] IV. Microfluidic Channels for Providing Constrictions to Deform Cells In some embodiments, the present invention provides a method for suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, the constriction being contained in a microfluidic channel that deforms anucleated cells so that antigens or compounds enter the anucleated cells, thereby causing perturbation of the cells. In some embodiments, multiple constrictions may be placed in parallel and / or in series in a microfluidic channel. An example of a microfluidic channel containing a constriction that deforms cells for use in the methods disclosed herein is described in WO2013059343. An example of a surface having holes for use in the methods disclosed herein is described in U.S. Provisional Application No. 62 / 214,820, filed September 4, 2015.

[0089] In some embodiments, the microfluidic channel comprises a lumen and is configured to allow cells suspended in a buffer to pass through, and the microfluidic channel comprises a constriction. The microfluidic channel can be made of any one of several materials, including silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene, PET, PETG), ceramics, glass, crystalline substrate, amorphous substrate, or polymer (e.g., polymethylmethacrylate (PMMA), PDMS, cyclic olefin copolymer (COC), etc.). Fabrication of the microfluidic channel can be performed by any method known in the art, including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 mask.

[0090] In some embodiments, the constriction in the microfluidic channel includes an inlet portion, a center point, and an outlet portion. In some embodiments, the length, depth, and width of the constriction in the microfluidic channel can vary. In some embodiments, the diameter of the constriction in the microfluidic channel is a function of the diameter of the cell or cell cluster. In some embodiments, the diameter of the constriction in the microfluidic channel is about 10% to about 99% of the diameter of the cell. In some embodiments, the constriction size is about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. In some embodiments, the constriction size is about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the smallest cross-sectional distance of the cell (e.g., an anuclear cell such as an RBC). In some embodiments, the channel comprises a constriction width of about 0.25 μm to about 4 μm. In some embodiments, the channel comprises a constriction width of about 4 μm, about 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1 μm, about 0.5 μm or about 0.25 μm (including any range between these values). Additionally, the cross-sections of the channel, the inlet portion, the center point and the outlet portion may vary. For example, the cross-section may be circular, elliptical, elongated slit, square, hexagonal or triangular in shape. The inlet section defines a constriction angle, which is optimized to reduce clogging of the channel and for improved delivery of compounds to the cells. The angle of the outlet section may also vary. For example, the angle of the outlet section is configured to reduce the likelihood of turbulence, which may result in non-laminar flow. In some embodiments, the walls of the inlet section and / or the outlet section are straight. In other embodiments, the walls of the inlet section and / or the outlet section are curved.

[0091] V. Surfaces with pores to provide constrictions for cell deformation In some embodiments, the present invention provides a method for suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, the constriction being a hole or contained within a hole, deforming the anucleated cells so that antigenic compounds enter the anucleated cells, thereby causing perturbation of the cells. In some embodiments, the hole is contained on a surface. Examples of surfaces with holes for use in the methods disclosed herein are described in U.S. Provisional Application No. 62 / 214,820, filed September 4, 2015.

[0092] The surfaces disclosed herein may be made from any one of several materials and may take any one of several forms. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface is a sponge or sponge-like matrix. In some embodiments, the surface is a matrix.

[0093] In some embodiments, the surface is a tortuous path surface. In some embodiments, the tortuous path surface comprises cellulose acetate. In some embodiments, the surface comprises a material selected from, but not limited to, synthetic or natural polymers, polycarbonate, silicon, glass, metals, alloys, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene, mixed cellulose esters, porcelain, and ceramic.

[0094] The surfaces disclosed herein can have any shape known in the art, including three-dimensional shapes. The two-dimensional shape of the surface can be, but is not limited to, a circle, an ellipse, a circle, a square, a star, a triangle, a polygon, a pentagon, a hexagon, a heptagon, or an octagon. In some embodiments, the surface is round in shape. In some embodiments, the three-dimensional shape of the surface is a cylinder, a cone, or a cube.

[0095] The surface can have a variety of cross-sectional widths and cross-sectional thicknesses. In some embodiments, the cross-sectional width of the surface is between about 1 mm and about 1 m, or any cross-sectional width or range of cross-sectional widths therebetween. In some embodiments, the surface has a defined thickness. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is variable. For example, in some embodiments, portions of the surface are thicker or thinner than other portions of the surface. In some embodiments, the thickness of the surface varies from about 1% to about 90%, or any percentage or range of thicknesses therebetween. In some embodiments, the surface is about 0.01 μm to about 5 mm thick, or any thickness or range of thicknesses therebetween.

[0096] In some embodiments, the constriction is a hole or is contained within a hole. The cross-sectional width of the hole is related to the type of cell being treated. In some embodiments, the hole size is a function of the diameter of the cell or cell of cluster of cells being treated. In some embodiments, the hole size is such that the cell is perturbed as it passes through the hole. In some embodiments, the hole size is less than the diameter of the cell. In some embodiments, the hole size is about 10% to about 99% of the diameter of the cell. In some embodiments, the hole size is about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter. The optimal hole size or cross-sectional width of the hole may vary based on the application and / or cell type. In some embodiments, the hole size is about 0.1 μm to about 4 μm. In some embodiments, the pore size is about 4 μm, about 3 μm, about 2 μm, about 1 μm, about 0.5 μm, about 0.25 μm, or about 0.1 μm. In some embodiments, the cross-sectional width of the pore is about 4 μm, about 3 μm, about 2 μm, about 1 μm, about 0.5 μm, about 0.25 μm, or about 0.1 μm, or less. The inlet and outlet of the pore passage may have various angles. The angle of the pore may be selected to minimize clogging of the pore during the passage of cells. In some embodiments, the flow rate through the surface is about 0.001 mL / cm 2 / sec ~ approx. 100L / cm 2 / sec or any speed or range of speeds therebetween. For example, the inlet or outlet portion angle can be between about 0 and about 90 degrees. In some embodiments, the inlet or outlet portion can be greater than 90 degrees. In some embodiments, the holes have the same inlet and outlet angles. In some embodiments, the holes have different inlet and outlet angles. In some embodiments, the edges of the holes are smooth, e.g., rounded, or curved. A smooth hole edge has a continuous, flat, and non-textured surface without bulges, bumps, or uneven portions. In some embodiments, the edges of the holes are sharp. A sharp hole edge has a sharp or sharp-angled thin edge. In some embodiments, the hole passage is straight. A straight hole passage does not have a curve, bend, corner, or other irregularity. In some embodiments, the hole passage is curved. A curved hole passage is bent or deviates from a straight line. In some embodiments, the pore passage has multiple curves, for example about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves.

[0097] The pores can have any shape known in the art, such as two-dimensional or three-dimensional shapes. The shape of the pores (e.g., the cross-sectional shape) may be, but is not limited to, circular, elliptical, round, square, star, triangle, polygon, pentagon, hexagon, heptagon, and octagon. In some embodiments, the cross-section of the pores is round in shape. In some embodiments, the three-dimensional shape of the pores is cylindrical or conical. In some embodiments, the pores have grooved inlet and outlet shapes. In some embodiments, the pore shapes are uniform (i.e., consistent or regular) among the pores in a given surface. In some embodiments, the pore shapes are non-uniform (i.e., mixed or variable) among the pores in a given surface.

[0098] The surfaces described herein can have a wide range of total pore numbers. In some embodiments, the pores range from about 10% to about 80% of the total surface area. In some embodiments, the surfaces have a total of about 1.0×10 5From 1.0×10 30 In some embodiments, the surface contains 1 mm 2 about 10 to about 1.0 × 10 per surface area of 15 It contains holes.

[0099] The pores can be distributed in a number of ways within a given surface. In some embodiments, the pores are distributed in parallel within a given surface. In one such example, the pores are distributed side by side in the same direction and are spaced apart the same distance within a given surface. In some embodiments, the distribution of the pores is regular or uniform. In one such example, the pores are distributed in a regular systematic pattern or are spaced apart the same distance within a given surface. In some embodiments, the distribution of the pores is random or non-uniform. In one such example, the pores are distributed in an irregular, chaotic pattern or are spaced apart different distances within a given surface. In some embodiments, multiple surfaces are distributed in rows. The multiple surfaces may be uniform or non-uniform in surface size, shape, and / or roughness. The multiple surfaces may further contain pores of uniform or non-uniform pore size, shape, and / or number, thereby allowing for the simultaneous delivery of a series of compounds to different cell types.

[0100] In some embodiments, individual pores have a uniform width dimension (i.e., a constant width along the length of the pore's path). In some embodiments, individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore's path). In some embodiments, pores within a given surface have the same individual pore depth. In some embodiments, pores within a given surface have different individual pore depths. In some embodiments, the pores are immediately adjacent to one another. In some embodiments, the pores are separated from one another by a distance. In some embodiments, the pores are separated from one another by a distance of about 0.001 μm to about 30 mm, or any distance or range of distances therebetween.

[0101] In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including, but not limited to, Teflon, adhesive coatings, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors modulating cell function, nucleic acids, fats, carbohydrates, or transmembrane proteins. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently attached to the surface. In some embodiments, the material is non-covalently attached to the surface. In some embodiments, the surface molecules are released when the cells pass through the pores.

[0102] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface may be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has an overall positive or overall negative charge. In some embodiments, the surface may be smooth, electropolished, rough, or plasma treated. In some embodiments, the surface comprises a zwitterionic or dipolar compound. In some embodiments, the surface is plasma treated.

[0103] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0104] VI. Cell perturbation In some embodiments, the present invention provides a method for suppressing an immune response or inducing tolerance by passing a cell suspension through a constriction, which deforms an anucleated cell such that an antigen or compound enters the anucleated cell, thereby causing a perturbation of the cell, the perturbation in the cell being a cleft (e.g., hole, lacuna, cavity, opening, hole, cleft, gap, perforation) in the cell that allows material from outside the cell to move into the cell. The deformation can be caused by, for example, mechanical strain and / or pressure induced by shear force. In some embodiments, the perturbation is a perturbation in the cell membrane. In some embodiments, the perturbation is transient. In some embodiments, the cell perturbation is about 1.0×10 -9 In some embodiments, the cellular perturbation lasts for about 1.0×10 seconds to about 2 hours, or any time or range of time therebetween. -9 In some embodiments, the cellular perturbation lasts for about 1.0×10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 ~Approx. 1.0×10 -1 , about 1.0×10 -9 ~Approx. 1.0×10 -2 , about 1.0×10 -9 ~Approx. 1.0×10 -3 , about 1.0×10 -9 ~Approx. 1.0×10 -4 , about 1.0×10 -9 ~Approx. 1.0×10 -5 , about 1.0×10 -9 ~Approx. 1.0×10 -6 , about 1.0×10 -9 ~Approx. 1.0×10 -7 or about 1.0 x 10 -9 ~Approx. 1.0×10 -8 In some embodiments, the cellular perturbation lasts for about 1.0×10 seconds. -8 ~Approx. 1.0×10 -1 , about 1.0×10 -7 ~Approx. 1.0×10 -1 , about 1.0×10 -6 ~Approx. 1.0×10 -1 , about 1.0×10 -5 ~Approx. 1.0×10 -1, about 1.0×10 -4 ~Approx. 1.0×10 -1 , about 1.0×10 -3 ~Approx. 1.0×10 -1 or about 1.0 x 10 -2 ~Approx. 1.0×10 -1 The cellular perturbations (e.g., holes or pores) created by the methods described herein are not formed as a result of assembly of protein subunits that form multimeric pore structures, such as those created by complement or bacterial hemolysin.

[0105] As the cells pass through the constriction, the constriction temporarily damages the cell membrane, which causes passive diffusion of material through the perturbation. In some embodiments, the cells are only deformed for a short period of time, on the order of 100 μs, to minimize the possibility of activating apoptotic pathways through cellular signaling mechanisms, although other durations are possible (e.g., ranging from a few nanoseconds to several hours). In some embodiments, the cells are ... on the order of 100 μs, although other durations are possible (e.g., ranging from a few nanoseconds to several hours). -9 In some embodiments, the cells are transformed for a period of time ranging from about 1.0×10 seconds to about 2 hours, or any period or range of time therebetween. -9 In some embodiments, the cells are deformed for about 1.0×10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -2 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -3 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -4 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -5 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -6 seconds, approximately 1.0×10 -9 seconds to about 1.0 x 10 -7 seconds, or approximately 1.0 x 10 -9 seconds to about 1.0 x 10 -8In some embodiments, the cells are deformed over a period of about 1.0×10 -8 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -7 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -6 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -5 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -4 seconds to about 1.0 x 10 -1 seconds, approximately 1.0×10 -3 seconds to about 1.0 x 10 -1 seconds, or approximately 1.0 x 10 -2 seconds to about 1.0 x 10 -1 In some embodiments, deforming the cells includes, but is not limited to, deforming the cells for a time ranging from about 1 μs to at least about 750 μs, such as at least about 1 μs, 10 μs, 50 μs, 100 μs, 500 μs, or 750 μs.

[0106] In some embodiments, passage of the compound into the cell occurs simultaneously with passage of the cell through the constriction and / or perturbation of the cell. In some embodiments, passage of the compound into the cell occurs after the cell has passed through the constriction. In some embodiments, passage of the compound into the cell occurs on the order of minutes ... about 1.0×10 20 30 40 50 60 70 80 90 100 150 160 180 190 200 350 200 400 500 600 700 800 900 1000 150 200 250 300 400 500 600 700 8 -2 For example, passage of the compound into the cell occurs about 1.0×10 seconds to at least about 30 minutes after the cell passes through the constriction. -2 In some embodiments, passage of the compound into the cell occurs about 1.0×10 s to about 1 s, about 1 s to about 1 min, or about 1 min to about 30 min after the cell has passed through the constriction. -2 seconds to about 10 minutes later, about 1.0 x 10 -2 seconds to about 5 minutes later, about 1.0 x 10 -2 seconds to about 1 minute, about 1.0 x 10-2 seconds to about 50 seconds later, about 1.0 x 10 -2 1.0×10 -2 seconds to about 1 second later, or about 1.0 x 10 -2 In some embodiments, passage of the compound into the cell occurs about 1.0×10 seconds to about 0.1 seconds after the cell passes through the constriction. -1 In some embodiments, the perturbation of the cell after passing through the constriction occurs within on the order of about 5 minutes after passing the cell through the constriction.

[0107] In some embodiments, the cell viability after passing through the constriction is about 5% to about 100%. In some embodiments, the cell viability after passing through the constriction is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the cell viability is about 1.0×10 after the cells pass through the constriction. -2 For example, the cell viability is measured at about 1.0×10 sec to at least about 10 days after the cells have passed through the constriction. -2 In some embodiments, the cell viability is measured after about 1.0×10 2 to about 1 second, about 1 second to about 1 minute, about 1 minute to about 30 minutes, or about 30 minutes to about 2 hours after the cells have passed through the constriction. -2 seconds to about 2 hours later, about 1.0 x 10 -2 seconds to about an hour later, about 1.0 x 10 -2 seconds to about 30 minutes later, about 1.0 x 10 -2 seconds to about 1 minute, about 1.0 x 10 -2 sec. to about 30 seconds later, about 1.0 x 10 -2 seconds to about 1 second later, or about 1.0 x 10 -2In some embodiments, the cell viability is measured about 1.5 hours to about 2 hours, about 1 hour to about 2 hours, about 30 minutes to about 2 hours, about 15 minutes to about 2 hours, about 1 minute to about 2 hours, about 30 seconds to about 2 hours, or about 1 second to about 2 hours after the cells have passed through the constriction. In some embodiments, the cell viability is measured about 2 hours to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 10 days after the cells have passed through the constriction.

[0108] VII. Delivery Parameters Several parameters may affect the delivery of compounds to cells to suppress immune responses or induce tolerance by the methods described herein. In some embodiments, the cell suspension is contacted with the compound before, simultaneously with, or after passing through the constriction. The cells may be suspended in a solution containing the compound to be delivered and passed through the constriction, or the compound may be added to the cell suspension after the cells have passed through the constriction. In some embodiments, the compound to be delivered is coated on the constriction.

[0109] Examples of parameters that may affect the delivery of the compound to the cells include, but are not limited to, the dimensions of the constriction, the entrance angle of the constriction, the surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), the operating flow speed (e.g., the passage time of cells through the constriction), the cell concentration, the concentration of the compound in the cell suspension, and the amount of time the cells recover or incubate after passing through the constriction may affect the passage of the compound delivered to the cells. Additional parameters that affect the delivery of the compound to the cells may include the velocity of the cells in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. Such parameters may be designed to control the delivery of the compound. In some embodiments, the cell concentration is between about 10 and at least about 10 12In some embodiments, the delivery compound concentration can range from about 10 ng / ml to about 1 g / mL or any concentration or concentration range therebetween. In some embodiments, the delivery compound concentration can range from about 1 pM to at least about 2M or any concentration or concentration range therebetween. The composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) can affect the delivery of the compound to suppress an immune response or induce tolerance. In some embodiments, the aqueous solution is isotonic or isotonic.

[0110] The temperature used in the method of the present disclosure can be adjusted to affect compound delivery and cell viability. In some embodiments, the method is carried out between about -5°C and about 45°C. For example, the method can be carried out at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., from about 37°C to 45°C or higher), or at reduced temperature (e.g., from about -5°C to about 4°C), or between these exemplary temperatures.

[0111] Various methods can be used to propel the cells through the constriction. For example, pressure can be applied to the inlet side by a pump (e.g., a gas cylinder or compressor), a vacuum can be applied to the outlet side by a vacuum pump, capillary action can be applied through tubing, and / or the system can be gravity fed. Displacement based flow systems can also be used (e.g., syringe pumps, peristaltic pumps, manual syringes or pipettes, pistons, etc.). In some embodiments, the cells are forced through the constriction by positive or negative pressure. In some embodiments, the cells are forced through the constriction by constant or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, pressure is applied using a vacuum. In some embodiments, the cells are forced through the constriction by gravity. In some embodiments, the cells are forced through the constriction by centrifugal force. In some embodiments, the cells are forced through the constriction by capillary pressure.

[0112] In some embodiments, the fluid flow directs the cells through the constriction. In some embodiments, the fluid flow is turbulent before the cells pass through the constriction. Turbulent flow is fluid flow with random variations in magnitude and direction of velocity at a given point. In some embodiments, the fluid flow through the constriction is laminar. Laminar flow involves a continuous flow of fluid near a solid boundary where the direction of flow remains constant at every point. In some embodiments, the fluid flow is turbulent after the cells pass through the constriction. The speed at which the cells pass through the constriction may be variable. In some embodiments, the cells pass through the constriction with a uniform cell velocity. In some embodiments, the cells pass through the constriction with a fluctuating cell velocity.

[0113] In other embodiments, a combination process, such as the methods described herein, followed by exposure to an electric field downstream of a constriction, is used to suppress an immune response or induce tolerance by passing a cell suspension through the constriction, which deforms anucleated cells such that antigens or compounds enter the anucleated cells, thereby causing perturbation of the cells. In some embodiments, the cells pass through an electric field generated by at least one electrode after passing through the constriction. In some embodiments, the electric field assists in the delivery of a compound to a second location within the cell. For example, the combination of a constriction that deforms a cell and an electric field delivers a plasmid encoding an antibody to the cell, resulting in de novo production of the antibody. In some embodiments, one or more electrodes are proximal to the constriction that deforms the cell and generate an electric field. In some embodiments, the electric field is between about 0.1 kV / m and about 100 MV / m, or any number or range of numbers therebetween. In some embodiments, an integrated circuit is used to provide the electrical signal that drives the electrodes. In some embodiments, the cells are exposed to the electric field for a pulse width between about 1 ns and about 1 s and a duration between about 100 ns and about 10 s, or any time or range of times therebetween.

[0114] VIII. Cell Suspensions for Delivery to Nonnucleated Cells The cell suspension may be a mixed or purified cell population. In some embodiments, the cell suspension is a mixed cell population, e.g., whole blood. In some embodiments, the cell suspension is a purified cell population, e.g., a purified population of anucleated cells.

[0115] The composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) may affect the delivery of the compound for suppressing an immune response or inducing tolerance. In some embodiments, the suspension comprises whole blood. Alternatively, the cell suspension is a mixture of cells in a physiological medium other than saline solution or blood. In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises cell culture medium, PBS, salts, sugars, growth factors, animal products, bulking materials, surfactants, lubricants, vitamins, amino acids, proteins, cell cycle inhibitors, and / or agents that affect actin polymerization. In some embodiments, the cell culture medium is DMEM, Opti-MEM™, IMDM, or RPMI. In addition, the solution buffer may comprise one or more lubricants (e.g., pluronics or other surfactants), which may be designed, for example, to reduce or eliminate clogging of the surface and improve cell viability. Examples of surfactants include, but are not limited to, poloxamers, polysorbates, sugars or sugar alcohols such as mannitol, sorbitol, animal derived serum, and albumin proteins. In some embodiments, the aqueous solution is isotonic or isotonic. In some embodiments, the aqueous solution comprises plasma.

[0116] In some configurations involving certain types of cells, the cells may be incubated in one or more solutions that aid in the delivery of the compound into the cells. In some embodiments, the aqueous solution includes an agent that affects actin polymerization. In some embodiments, the agent that affects actin polymerization is Latrunculin A, cytochalasin, and / or colchicine. For example, the cells can be incubated in a depolymerizing solution such as Lantrunculin A (0.1 μg / ml) for 1 hour prior to delivery to depolymerize the actin cytoskeleton. As an additional example, the cells can be incubated in 10 μM colchicine (Sigma) for 2 hours prior to delivery to depolymerize the microtubule network.

[0117] In some embodiments, the cell population is enriched before being used in the disclosed method.For example, cells are obtained from body fluids (e.g., peripheral blood), and then enriched or purified as necessary to enrich B cells.Cells can be enriched by any method known in the art, such as, but not limited to, magnetic cell separation, fluorescence-activated cell sorting (FACS), or density gradient centrifugation.

[0118] The viscosity of the cell suspension may also affect the methods disclosed herein. In some embodiments, the viscosity of the cell suspension is about 8.9×10 -4 Pa.s to approx. 4.0×10 -3 In some embodiments, the viscosity is about 8.9×10 Pa.s or any value or range of values ​​therebetween. -4 Pa.s to approx. 4.0×10 -3 During Pa.s, approximately 8.9 × 10 -4 Pa.s to approx. 3.0×10 -3 During Pa.s, approximately 8.9 × 10 -4 Pa.s to approx. 2.0×10 -3 Between Pa.s, or about 8.9 x 10 -3 Pa.s to approx. 1.0×10 -3In some embodiments, the viscosity ranges from about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about 0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some embodiments, a shear-thinning effect is observed, and the viscosity of the cell suspension decreases under conditions of shear strain. Viscosity can be measured by any method known in the art, including, but not limited to, a viscometer, such as a glass capillary viscometer, or a rheometer. A viscometer measures viscosity under one flow condition, while a rheometer is used to measure viscosity under changing flow conditions. In some embodiments, the viscosity is measured for a shear-thinning solution, such as blood. In some embodiments, the viscosity is measured between about -5°C and about 45°C. For example, the viscosity is measured at room temperature (e.g., about 20° C.), physiological temperature (e.g., about 37° C.), above physiological temperature (e.g., from about above 37° C. to 45° C. or higher), or at reduced temperature (e.g., from about −5° C. to about 4° C.), or at a temperature between these exemplary temperatures.

[0119] IX. Antigens and Tolerogenic Factors for Suppressing Immune Responses or Inducing Tolerance In some embodiments, the present invention provides for the delivery of an antigen to suppress immune responses or induce tolerance, wherein the antigen is delivered to the cells by any of the methods described herein. In some embodiments, the antigen is a single antigen. In some embodiments, the antigen is a mixture of antigens. An antigen is a substance that stimulates a specific immune response, such as a cell- or antibody-mediated immune response. An antigen binds to a receptor expressed by an immune cell that is specific for a particular antigen, such as a T cell receptor (TCR). The binding of the antigen to the receptor subsequently triggers intracellular signaling pathways that lead to downstream immune effector pathways, such as cell activation, cytokine production, cell migration, cytotoxic factor secretion, cell apoptosis, and antibody production.

[0120] In some embodiments, the antigen is a protein or polypeptide antigen. In some embodiments, the compound comprises a disease-associated antigen. In some embodiments, the antigen is from an exogenous source, such as a bacterium, fungus, virus, or allergen. In some embodiments, the antigen is from an internal source, such as a self-protein (i.e., an autoantigen). In some embodiments, the antigen is in a cell lysate. An autoantigen is an antigen present on or within an organism's own cells. Autoantigens do not normally stimulate an immune response, but may in the context of autoimmune disease, such as type I diabetes or rheumatoid arthritis. In some embodiments, the antigen is a therapeutic agent. In some embodiments, the antigen is a therapeutic polypeptide or a fragment of a therapeutic polypeptide. In some embodiments, the therapeutic agent is conjugated to a carbohydrate. In some embodiments, the therapeutic agent is a clotting factor. Examples of clotting factors include, but are not limited to, factor VIII and factor IX. For example, the antigen may be factor VIII protein, which is used in the treatment of hemophilia. In some embodiments, the therapeutic agent is an antibody. Examples of therapeutic antibodies include, but are not limited to, anti-TNFα, anti-VEGF, anti-CD3, anti-CD20, anti-IL-2R, anti-Her2, anti-RSVF, anti-CEA, anti-IL-1 beta, anti-CD15, anti-myosin, anti-PSMA, anti-40kDa glycoprotein, anti-CD33, anti-CD52, anti-IgE, anti-CD11a, anti-EGFR, anti-C5, anti-alpha-4 integrin, anti-IL-12 / IL-23, anti-IL-6R, and anti-RANKL. In some embodiments, the therapeutic agent is a growth factor. Examples of therapeutic growth factors include, but are not limited to, erythropoietin (EPO) and megakaryocyte differentiation growth factor (MDGF). In some embodiments, the therapeutic agent is a hormone. Examples of therapeutic hormones include, but are not limited to, insulin, human growth hormone, and follicle stimulating hormone. In some embodiments, the therapeutic agent is a recombinant cytokine. Examples of therapeutic recombinant cytokines include, but are not limited to, IFNβ, IFNα, and GM-CSF.

[0121] In some embodiments, the antigen is an allograft antigen. Allografting is the transfer of cells, tissues or organs from a genetically non-identical donor of the same species to a recipient. In some embodiments, the antigen is a modified antigen. For example, the antigen may be fused to a therapeutic agent or a targeting peptide. In some embodiments, the modified antigen is fused to a polypeptide. In some embodiments, the modified antigen is fused to a lipid. In some embodiments, the antigen is fused to a carbohydrate. In some embodiments, the antigen is a non-protein antigen, such as a lipid, glycolipid or polysaccharide. In some embodiments, the antigen is a whole microorganism, such as an intact bacterium.

[0122] In some embodiments, the antigen is associated with a virus. In some embodiments, the antigen is a viral antigen. Exemplary viral antigens include SARS-CoV antigens and influenza antigens. In some embodiments, the compound comprises a cell lysate from a tissue infected with an unknown pathogen. In some embodiments, the antigen is a therapeutic vehicle. In some embodiments, the therapeutic vehicle is a virus, such as an adenovirus, an adeno-associated virus, a baculovirus, a herpes virus, or a retrovirus, used for gene therapy. In some embodiments, the therapeutic vehicle is a liposome. In some embodiments, the therapeutic vehicle is a nanoparticle.

[0123] In some embodiments, the antigen is associated with a microbe (e.g., a bacterium). In some embodiments, the suppressed immune response and / or induced tolerance comprises a reduced pathogenic immune response or induced tolerance to the microbe (e.g., a bacterium). In some embodiments, the microbe is part of the individual's microbiome. In some embodiments, the microbe may provide a benefit to the individual's microbiome.

[0124] In certain aspects, the invention provides a method for delivering an antigen to an anucleated cell, comprising passing a cell suspension comprising the anucleated cells through a constriction, the constriction deforming the anucleated cells such that the antigen enters the cells, thereby causing perturbation of the cells, where the cell suspension contacts the antigen. In some embodiments, presentation of the antigen in a tolerogenic environment suppresses an immune response to the antigen. In some embodiments, presentation of the antigen in a tolerogenic environment induces tolerance to the antigen. In some embodiments, the antigen is delivered to the anucleated cells in vitro, ex vivo, or in vivo.

[0125] In some embodiments, the invention provides for delivery of a tolerogenic factor to suppress an immune response or induce tolerance, wherein the tolerogenic factor is delivered to the cell by any of the methods described herein. In some embodiments, the tolerogenic factor improves suppression of an immune response to an antigen and / or improves induction of tolerance to an antigen. For example, the tolerogenic factor may promote tolerogenic presentation of the antigen by an antigen presenting cell. In some embodiments, the tolerogenic factor is introduced simultaneously with the antigen. In some embodiments, the tolerogenic factor and antigen are introduced sequentially. In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα, or TGFβ. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, the polypeptide is conjugated to a carbohydrate. In some embodiments, the tolerogenic factor is a nucleic acid. In some embodiments, the nucleic acid can include, but is not limited to, mRNA, DNA, miRNA or siRNA.For example, the tolerogenic agent can include siRNA for knocking down the expression of inflammatory genes.In some embodiments, the tolerogenic agent is a DNA sequence that binds to NFκB and prevents NFκB activation and downstream signal transduction.In some embodiments, the tolerogenic agent is a small molecule.

[0126] In some embodiments, the tolerogenic factor modulates the expression and / or activity of an immunomodulatory factor, such as, for example, an immunostimulatory factor (e.g., a costimulatory molecule), an immunosuppressant, or an inflammatory or anti-inflammatory molecule. In some embodiments, the tolerogenic factor inhibits the expression and / or activity of an immunostimulatory factor (e.g., a costimulatory molecule), enhances the expression and / or activity of an immunosuppressant molecule, inhibits the expression and / or activity of an inflammatory molecule, and / or enhances the expression and / or activity of an anti-inflammatory molecule. In some embodiments, the tolerogenic factor inhibits the activity of a costimulatory molecule. The interaction between costimulatory molecules and their ligands is important to maintain and integrate TCR signaling to stimulate optimal T cell proliferation and differentiation. In some embodiments, the tolerogenic factor reduces the expression of a costimulatory molecule. Exemplary costimulatory molecules expressed on antigen presenting cells include, but are not limited to, CD40, CD80, CD86, CD54, CD83, CD79, or ICOS ligand. In some embodiments, the costimulatory molecule is CD80 or CD86. In some embodiments, the tolerogenic factor inhibits expression of a nucleic acid that expresses or modulates the expression of the costimulatory molecule. In some embodiments, the tolerogenic factor deletes a nucleic acid that expresses or modulates the expression of the costimulatory molecule. In some embodiments, deletion of the nucleic acid that expresses or modulates the expression of the costimulatory molecule is accomplished through gene editing. In some embodiments, the tolerogenic factor inhibits the costimulatory molecule. In some embodiments, the tolerogenic factor is an siRNA that inhibits the costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that suppresses the expression of the costimulatory molecule. In some embodiments, the tolerogenic factor increases the activity of a protein inhibitor that suppresses the expression of a costimulatory molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a suppressor of the costimulatory molecule. In some embodiments, the tolerogenic factor degrades the costimulatory molecule. In some embodiments, the tolerogenic factor tags the costimulatory molecule for destruction.For example, tolerogenic factors may enhance the ubiquitination of the costimulatory molecule, thereby targeting it for destruction.

[0127] In some embodiments, the tolerogenic factor enhances the expression and / or activity of an immunosuppressive molecule. In some embodiments, the immunosuppressive molecule is a co-inhibitory molecule, a transcriptional regulator, or an immunosuppressive molecule. A co-inhibitory molecule negatively regulates lymphocyte activation. Exemplary co-inhibitory molecules include, but are not limited to, PD-L1, PD-L2, HVEM, B7-H3, TRAIL, immunoglobulin-like transcript (ILT) receptors (ILT2, ILT3, ILT4), FasL, CTLA4, CD39, CD73, and B7-H4. In some embodiments, the co-inhibitory molecule is PD-L1 or PD-L2. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the expression of a co-inhibitory molecule. In some embodiments, the tolerogenic factor encodes the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances expression of the co-inhibitory molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that increases expression of the co-inhibitory molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of the co-inhibitory molecule. In some embodiments, the tolerogenic factor inhibits an inhibitor of a co-inhibitory molecule.

[0128] In some embodiments, the tolerogenic factor increases the expression and / or activity of an immunosuppressive molecule. Exemplary immunosuppressive molecules include, but are not limited to, arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), nitric oxide synthase 2 (NOS2), thymic stromal lymphopoietin (TSLP), vasoactive intestinal peptide (VIP), hepatocyte growth factor (HGF), transforming growth factor beta (TGFβ), IFNα, IL-4, IL-10, IL-13, and IL-35. In some embodiments, the immunosuppressive molecule is NO or IDO. In some embodiments, the tolerogenic factor encodes the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a transcriptional regulator that enhances expression of the immunosuppressive molecule. In some embodiments, the tolerogenic factor increases the activity of a polypeptide that enhances expression of the immunosuppressive molecule. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of the immunosuppressive molecule. In some embodiments, the tolerogenic factor inhibits a negative regulator of the immunosuppressive molecule.

[0129] In some embodiments, the tolerogenic factor inhibits the expression and / or activity of an inflammatory molecule. In some embodiments, the inflammatory molecule is an inflammatory transcription factor. In some embodiments, the tolerogenic factor inhibits the inflammatory transcription factor. In some embodiments, the tolerogenic factor reduces the expression of an inflammatory transcription factor. In some embodiments, the inflammatory transcription factor is a molecule associated with the NF-κB, interferon regulatory factor (IRF), or JAK-STAT signaling pathway. The NF-κB pathway is the prototypical proinflammatory signaling pathway that mediates the expression of proinflammatory genes such as cytokines, chemokines, and adhesion molecules. Interferon regulatory factors (IRFs) constitute a family of transcription factors that can regulate the expression of proinflammatory genes. The JAK-STAT signaling pathway transmits information from extracellular cytokine signals to the nucleus, resulting in DNA transcription and expression of genes involved in the proliferation and differentiation of immune cells. The JAK-STAT system consists of cell surface receptors, Janus kinases (JAKs), and signal transducers and activators of transcription (STATs) proteins. Exemplary JAK-STAT molecules include, but are not limited to, JAK1, JAK2, JAK3, Tyk2, STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6. In some embodiments, the tolerogenic factor enhances expression of suppressors of cytokine signaling (SOCS) proteins. SOCS proteins can inhibit signaling through the JAK-STAT pathway. In some embodiments, the tolerogenic factor inhibits expression of a nucleic acid encoding the inflammatory transcription factor. In some embodiments, the tolerogenic factor deletes a nucleic acid encoding the inflammatory transcription factor. In some embodiments, the tolerogenic factor increases activity of a transcription regulator that suppresses expression of the inflammatory transcription factor. In some embodiments, the tolerogenic factor increases activity of a protein inhibitor that suppresses expression of the inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a suppressor of the inflammatory transcription factor.

[0130] In some embodiments, the tolerogenic factor enhances expression and / or activity of an anti-inflammatory molecule. In some embodiments, the anti-inflammatory molecule is an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor increases expression of an anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor enhances expression of a nucleic acid encoding the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases activity of a transcription regulator that inhibits expression of the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor decreases activity of a protein inhibitor that inhibits expression of the anti-inflammatory transcription factor. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an enhancer of the anti-inflammatory transcription factor.

[0131] In certain aspects, the invention provides a method for delivering a tolerogenic factor to an anucleated cell, comprising passing a cell suspension comprising the anucleated cells through a constriction, the constriction deforming the anucleated cells such that the tolerogenic factor enters the cells, thereby causing perturbation of the cells, where the cell suspension is contacted with the tolerogenic factor. In some embodiments, the tolerogenic factor is delivered to the anucleated cells in vitro, ex vivo, or in vivo.

[0132] In some embodiments, the tolerogenic factor comprises a nucleic acid. In some embodiments, the tolerogenic factor is a nucleic acid. Exemplary nucleic acids include, but are not limited to, recombinant nucleic acid, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, guide RNA, and shRNA. In some embodiments, the nucleic acid is homologous to the nucleic acid in the cell. In some embodiments, the nucleic acid is heterologous to the nucleic acid in the cell. In some embodiments, the tolerogenic factor is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding an siRNA, mRNA, miRNA, lncRNA, tRNA, or shRNA. For example, the tolerogenic factor may comprise an siRNA for knocking down expression of an inflammatory gene. In some embodiments, the tolerogenic factor is a DNA sequence that binds to NFκB and prevents activation of NFκB and downstream signaling.

[0133] In some embodiments, the tolerogenic factor comprises a polypeptide. In some embodiments, the tolerogenic factor is a polypeptide. In some embodiments, the protein or polypeptide is a therapeutic protein, an antibody, a fusion protein, an antigen, a synthetic protein, a reporter marker, or a selectable marker. In some embodiments, the protein is a gene editing protein or a nuclease, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a CRE recombinase, a transposase, an RNA-guided endonuclease (e.g., CAS9 enzyme), a DNA-guided endonuclease, or an integrase enzyme. In some embodiments, the fusion protein can include, but is not limited to, a chimeric protein drug, such as an antibody drug conjugate, or a recombinant fusion protein, such as a protein tagged with GST or streptavidin. In some embodiments, the compound is a transcription factor. Examples of transcription factors include, but are not limited to, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORγt. In some embodiments, the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα, or TGFβ. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is a fragment of a therapeutic polypeptide. In some embodiments, the polypeptide is a peptide nucleic acid (PNA).

[0134] In some embodiments, the tolerogenic factor comprises a protein-nucleic acid complex. In some embodiments, the tolerogenic factor is a protein-nucleic acid complex. In some embodiments, protein-nucleic acid complexes such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 are used in genome editing applications. These complexes contain a sequence-specific DNA binding domain in combination with a non-specific DNA cleavage nuclease. These complexes allow targeted genome editing, such as adding, destroying, or changing the sequence of a specific gene. In some embodiments, disabled CRISPR is used to block or induce transcription of a target gene. In some embodiments, the tolerogenic factor comprises a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the tolerogenic factor comprises a nucleic acid encoding a Cas9 protein and a guide RNA or donor DNA. In some embodiments, the gene editing complex targets the expression of a costimulatory molecule (e.g., CD80 and / or CD86).

[0135] In some embodiments, the tolerogenic agent comprises a chimeric antigen receptor (CAR). In some embodiments, the tolerogenic agent is a chimeric antigen receptor (CAR). In some embodiments, the CAR is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding, the intracellular signaling portion of the CAR can initiate an immune suppressive or tolerogenic associated response in the immune cell. In some embodiments, the CAR is a chimeric T cell antigen receptor. In some embodiments, the antigen-binding domain of the CAR is a single chain antibody variable fragment (scFv). In some embodiments, the tolerogenic agent encodes a modified TCR that contains a cytoplasmic signaling domain that induces the production of immunosuppressive cytokines upon antigen binding. In some embodiments, the tolerogenic agent encodes a chimeric antigen receptor that contains a cytoplasmic signaling domain that induces the production of immunosuppressive cytokines upon antigen binding.

[0136] In some embodiments, the tolerogenic factor comprises a small molecule. In some embodiments, the tolerogenic factor is a small molecule. In some embodiments, the small molecule inhibits the activity of a costimulatory molecule, enhances the activity of a co-inhibitory molecule, and / or inhibits the activity of an inflammatory molecule. Examples of small molecules include, but are not limited to, pharmaceutical agents, metabolites, or radionuclides. In some embodiments, the pharmaceutical agent is a therapeutic drug and / or a cytotoxic agent. In some embodiments, the compound comprises a nanoparticle. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendrimers, and liposomes. In some embodiments, the nanoparticle contains a therapeutic molecule or is linked (covalently or non-covalently) to a therapeutic molecule. In some embodiments, the nanoparticle contains a nucleic acid, such as mRNA or cDNA.

[0137] In some embodiments, the compound delivered is purified. In some embodiments, the compound is at least about 60% by weight (dry weight) of the compound of interest. In some embodiments, the purified compound is at least about 75%, 90%, or 99% of the compound of interest. In some embodiments, the purified compound is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the compound of interest. Purity is determined by any known method, including but not limited to, column chromatography, thin layer chromatography, HPLC analysis, NMR, mass spectrometry, or SDS-PAGE. Purified DNA or RNA is defined as DNA or RNA that is free of exogenous nucleic acids, carbohydrates, and fats.

[0138] In some embodiments, the compound is an intermediate compound. The intermediate compound may be a molecular entity formed from a preceding intermediate and further reacted to give a final reaction product. In some embodiments, the intermediate compound is a protein precursor, or proprotein, that is cleaved by an enzyme to produce a mature, functional form of a protein. In some embodiments, the intermediate compound is an inactive enzyme precursor, or zymogen, that requires modification or cleavage to produce an active enzyme.

[0139] X.Apply In some aspects, the present invention provides a method of treating a patient by introducing modified non-nucleated cells into the patient by passing the modified non-nucleated cells through a constriction such that the compound enters the non-nucleated cells. In some embodiments, the treatment comprises multiple (e.g., 2, 3, 4, 5, 6 or more) steps of introducing such modified non-nucleated cells into the patient. In some embodiments, the cells are isolated from the patient, modified according to the disclosed methods, and introduced back into the patient. For example, a non-nucleated cell population is isolated from the patient, passed through the constriction to achieve delivery of a compound, and then re-infused into the patient to enhance a therapeutic immune response. In some embodiments, the cells are isolated from an individual, modified according to the disclosed methods, and introduced back into the individual. For example, a non-nucleated cell population is isolated from an individual, passed through the constriction to achieve delivery of a compound, and then re-infused into the patient to suppress an immune response or induce tolerance in the individual.

[0140] In some embodiments, anucleated cells are isolated from a universal donor (e.g., a type O donor) and then stored and / or frozen for later stenosis-mediated delivery and introduction into an individual. In some embodiments, antigens are isolated from an individual, delivered to anucleated cells isolated from a universal donor, and introduced into the individual. In some embodiments, anucleated cells are isolated from a donor and then stored and / or frozen for later stenosis-mediated delivery and introduction into an individual of a blood type compatible with the donor. In some embodiments, antigens are isolated from an individual, delivered to anucleated cells isolated from a donor, and introduced into the individual.

[0141] In some embodiments, the present invention provides a method of treating an individual by introducing into the individual cells that have been modified by passing the cells through a constriction to allow the compound to enter the cells. In some embodiments, the cells are autologous cells. For example, the anucleated cells are isolated from an individual (e.g., a patient), modified according to the disclosed methods, and introduced back into the individual. In some embodiments, the cells are isolated from an individual, modified according to the disclosed methods, and introduced back into the same individual. In some embodiments, the cells are allogeneic cells. For example, the cells are isolated from a different individual, modified according to the disclosed methods, and introduced into a first individual (e.g., a patient). In some embodiments, a pool of cells from multiple individuals is modified according to the disclosed methods and introduced into the first individual (e.g., the patient). In some embodiments, the cells are isolated from an individual, modified according to the disclosed methods, and introduced into a different individual. In some embodiments, a population of cells is isolated from an individual (a patient) or a different individual, passed through the constriction to achieve delivery of a compound, and then reinfused into the patient to enhance the therapeutic response.

[0142] In some embodiments, the treatment comprises multiple (e.g., 2, 3, 4, 5, 6 or more) steps of administering to the individual anucleated cells modified as described herein. For example, in some embodiments, a method is provided for treating the individual by administering to the individual 2, 3, 4, 5, 6 or more times the cells modified by passing through a constriction so that the compound enters the cells. In some embodiments, the time interval between any two consecutive administrations of the cells is at least about 1 day (e.g., at least about 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more, including any ranges between these values).

[0143] Any of the above methods may be performed in vitro, ex vivo, or in vivo. For in vivo applications, a device may be implanted into the lumen of a blood vessel, for example, an in-line stent may be implanted into an artery or vein. In some embodiments, the method is used as part of a bedside system for ex-vivo processing of patient cells and immediate reintroduction of the cells into the patient. Such methods may be used as a means to suppress immune responses or induce tolerance in individuals. In some embodiments, the method may be performed in a typical hospital laboratory with minimally trained technicians. In some embodiments, a patient operated treatment system may be used. In some embodiments, the method is performed using an in-line blood processing system, in which blood is drawn directly from a patient, passed through the stenosis to effect compound delivery to blood cells, and transfused directly back into the patient after processing.

[0144] XI. Systems and Kits In some aspects, the present invention provides a system comprising the constriction, a cell suspension and a compound for use in the methods disclosed herein. The system may include any embodiment described in the methods disclosed above, including a surface with microfluidic channels or holes to provide a constriction that transforms cells, a cell suspension, cell perturbation, delivery parameters, compounds and / or applications, etc. In some embodiments, the constriction that transforms cells is sized for delivery to anucleated cells. In some embodiments, the delivery parameters, such as operating flow rate, cell and compound concentration, the velocity of the cells in the constriction, and the composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.), are optimized for maximum response of compounds to suppress immune responses or induce tolerance.

[0145] Also provided are kits or articles for use in delivering compounds for suppressing immune responses or inducing tolerance. In some embodiments, the kits include compositions described herein (e.g., surfaces containing microfluidic channels or holes, cell suspensions, and / or compounds) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These products may be further sterilized and / or sealed.

[0146] The present disclosure also provides kits that include the components of the methods described herein and may further include instruction(s) for carrying out the methods to suppress an immune response or induce tolerance. The kits described herein may further include other materials, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for carrying out any of the methods described herein, such as for suppressing an immune response or inducing tolerance.

[0147] XII. Illustrative Embodiments Embodiment 1. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0148] Embodiment 2. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the tolerogenic factor contributes to a tolerogenic environment that suppresses the immune response.

[0149] Embodiment 3. A method for suppressing an immune response in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigens and tolerogenic factors enter the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0150] Embodiment 4. A method for suppressing an immune response in an individual, comprising: a. passing a first cell suspension containing first non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the non-nucleated cells; b. passing a second cell suspension containing second non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the non-nucleated cells; c. introducing the first non-nucleated cell and the second non-nucleated cell into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0151] Embodiment 5. A method for suppressing an immune response in an individual comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises an antigen, wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing perturbation of the cell, wherein the antigen is processed in a tolerogenic environment, and wherein presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0152] Embodiment 6. A method for suppressing an immune response in an individual comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises an antigen and a tolerogenic factor, wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing perturbation of the cell, wherein the antigen is processed in a tolerogenic environment, and wherein presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen.

[0153] Embodiment 7. A method for delivering a tolerogenic factor to anucleated cells, comprising passing a cell suspension comprising said anucleated cells through a constriction, said constriction deforming said anucleated cells such that said tolerogenic factor enters said cells, thereby causing perturbation of said cells, wherein said cell suspension is contacted with said tolerogenic factor.

[0154] Embodiment 8. A method for introducing an antigen into a tolerogenic environment comprising the step of delivering to an individual an anucleated cell, wherein the anucleated cell contains an antigen, and wherein the antigen is introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen enters the anucleated cell, thereby causing a perturbation of the cell, and wherein the antigen is processed in the tolerogenic environment.

[0155] Embodiment 9. A method for introducing an antigen and a tolerogenic factor into a tolerogenic environment comprising the step of delivering to an individual an anucleated cell, wherein the anucleated cell comprises the antigen and the tolerogenic factor, and wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing a perturbation of the cell, and wherein the antigen is processed in the tolerogenic environment.

[0156] Embodiment 10. A method for inducing antigen-specific tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0157] Embodiment 11. A method for inducing tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the tolerogenic factor contributes to a tolerogenic environment that induces tolerance.

[0158] Embodiment 12. A method for inducing tolerance in an individual, comprising: a. passing a cell suspension containing anucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigens and tolerogenic factors enter the anucleated cells; b. introducing the non-nucleated cells into the individual; wherein the antigen is processed in a tolerogenic environment and presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0159] Embodiment 13. A method for inducing antigen-specific tolerance in an individual, comprising: a. passing a first cell suspension containing first non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that antigen enters the non-nucleated cells; b. passing a second cell suspension containing second non-nucleated cells through a constriction, the constriction deforming the cells and thereby perturbing the cells such that the tolerogenic factor enters the non-nucleated cells; c. introducing the first non-nucleated cell and the second non-nucleated cell into the individual; wherein said antigen and said tolerogenic factor are processed in a tolerogenic environment, and presentation of said antigen in said tolerogenic environment induces tolerance to said antigen.

[0160] Embodiment 14. A method for inducing antigen-specific tolerance in an individual comprising the step of introducing into said individual anucleated cells, wherein said anucleated cells contain an antigen, said antigen being introduced into said anucleated cells by passing said anucleated cells through a constriction, wherein said constriction deforms said cells such that said antigen enters said anucleated cells, thereby causing perturbation of said cells, said antigen being processed in a tolerogenic environment, and presentation of said antigen in said tolerogenic environment induces tolerance to said antigen.

[0161] Embodiment 15. A method for inducing antigen-specific tolerance in an individual comprising the step of introducing an anucleated cell into the individual, wherein the anucleated cell comprises an antigen and a tolerogenic factor, wherein the antigen and the tolerogenic factor are introduced into the anucleated cell by passing the anucleated cell through a constriction, wherein the constriction deforms the cell such that the antigen and the tolerogenic factor enter the anucleated cell, thereby causing perturbation of the cell, wherein the antigen is processed in a tolerogenic environment, and wherein presentation of the antigen in the tolerogenic environment induces tolerance to the antigen.

[0162] Embodiment 16. A method for delivering an antigen to an anucleated cell, comprising passing a cell suspension containing the anucleated cells through a constriction, the constriction deforming the anucleated cells and thereby causing perturbation of the cells such that the antigen enters the cells, wherein the cell suspension comes into contact with the antigen.

[0163] Embodiment 17. A method for delivering an antigen and a tolerogenic factor to an anucleated cell, comprising passing a cell suspension comprising said anucleated cells through a constriction, said constriction deforming said anucleated cells such that said antigen and said tolerogenic factor enter said cells, thereby causing perturbation of said cells, wherein said cell suspension comes into contact with said antigen and said tolerogenic factor.

[0164] Embodiment 18 The method of embodiment 16 or 17, wherein processing and presentation of the antigen in a tolerogenic environment suppresses the immune response to the antigen.

[0165] Embodiment 19. The method of embodiment 16 or 17, wherein processing and presentation of said antigen in a tolerogenic environment induces tolerance to said antigen.

[0166] Embodiment 20. The method of any one of embodiments 1, 4, 5, 8, 10 and 13-19, wherein at least one additional antigen is introduced into the cells.

[0167] Embodiment 21. The method of any one of embodiments 2-4, 6, 7, 9, 11, 12, 13, 15 and 17, wherein at least one additional tolerogenic factor is introduced into the cells.

[0168] Embodiment 22. The method of any one of embodiments 1-6, 8-15, 18 and 19, wherein the tolerogenic environment is located in the spleen, liver or lymph nodes.

[0169] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the constriction is contained within a microfluidic channel.

[0170] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the channel comprises a constriction width of about 0.25 μm to about 4 μm.

[0171] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the channel comprises a constriction width of about 4 μm, 3.5 μm, about 3 μm, about 2.5 μm, about 2 μm, about 1.5 μm, about 1 μm, about 0.5 μm or about 0.25 μm.

[0172] Embodiment 26. The method of any one of embodiments 1 to 22, wherein the constriction is a hole or is contained within a hole.

[0173] Embodiment 27 The method of embodiment 26, wherein the pores are contained on the surface.

[0174] Embodiment 28 The method of embodiment 27, wherein the surface is a filter.

[0175] Embodiment 29 The method of embodiment 27, wherein the surface is a membrane.

[0176] Embodiment 30. The method of any one of embodiments 26 to 29, wherein the pore size is from about 0.5 μm to about 4 μm.

[0177] Embodiment 31. The method of any one of embodiments 26 to 30, wherein the pore size is 4 μm, about 3 μm, about 2 μm, about 1 μm or about 0.5 μm.

[0178] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the constriction size is a function of the diameter of the anucleated cells.

[0179] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70% of the cell diameter.

[0180] Embodiment 34. The method of any one of embodiments 1 to 33, wherein the method is carried out at between about -5°C and about 45°C.

[0181] Embodiment 35. The method of any one of embodiments 1-4, 7, 10-13, and 16-34, wherein the cell suspension comprises a mixed population of cells.

[0182] Embodiment 36 The method of embodiment 35, wherein the cell suspension is whole blood.

[0183] Embodiment 37. The method of any one of embodiments 1-4, 7, 10-13 and 16-34, wherein the cell suspension comprises a purified cell population.

[0184] Embodiment 38 The method of embodiment 37, wherein the cell suspension comprises a purified, non-nucleated cell population.

[0185] Embodiment 39. The method of any one of embodiments 1-4, 7, 10-13 and 16-38, wherein the cell suspension comprises mammalian cells.

[0186] Embodiment 40. The method of any one of embodiments 1-4, 7, 10-13 and 16-39, wherein the cell suspension comprises monkey cells, mouse cells, dog cells, cat cells, horse cells, rat cells, sheep cells, goat cells, pig cells or rabbit cells.

[0187] Embodiment 41. The method of any one of embodiments 1-4, 7, 10-13 and 16-39, wherein the cell suspension comprises human cells.

[0188] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the non-nucleated cells are red blood cells.

[0189] Embodiment 43. The method of embodiment 42, wherein the red blood cell is an erythrocyte.

[0190] Embodiment 44 The method of embodiment 42, wherein the red blood cells are reticulocytes.

[0191] Embodiment 45. The method of any one of embodiments 1 to 41, wherein the non-nucleated cells are platelets.

[0192] Embodiment 46. The method of any one of embodiments 1 to 45, wherein the non-nucleated cells are mammalian cells.

[0193] Embodiment 47. The method of any one of embodiments 1 to 46, wherein the anuclear cell is a monkey cell, a mouse cell, a dog cell, a cat cell, a horse cell, a rat cell, a sheep cell, a goat cell, a pig cell or a rabbit cell.

[0194] Embodiment 48. The method of any one of embodiments 1 to 46, wherein the anucleated cells are human cells.

[0195] Embodiment 49. The method of any one of embodiments 1-5, 8-14 and 22-48, wherein the anucleated cells are derived from the individual.

[0196] Embodiment 50. The method of any one of embodiments 1-5, 8-14 and 22-48, wherein the anucleated cells are derived from a different individual.

[0197] Embodiment 51. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is in a cell lysate.

[0198] Embodiment 52. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a foreign antigen.

[0199] Embodiment 53. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is an autoantigen.

[0200] Embodiment 54. The method of any one of embodiments 1, 4, 5, 8-10, and 13-50, wherein the antigen is an allograft antigen.

[0201] Embodiment 55. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a protein or polypeptide.

[0202] Embodiment 56. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a lysate.

[0203] Embodiment 57. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a microorganism.

[0204] Embodiment 58. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a lipid antigen.

[0205] Embodiment 59. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is a carbohydrate antigen.

[0206] Embodiment 60. The method of any one of embodiments 1, 4, 5, 8-10 and 13-59, wherein the antigen is a modified antigen.

[0207] Embodiment 61 The method of embodiment 60, wherein the modified antigen comprises an antigen fused to a polypeptide.

[0208] Embodiment 62 The method of embodiment 60 or 61, wherein the modified antigen comprises an antigen fused to a therapeutic agent.

[0209] Embodiment 63 The method of embodiment 60 or 61, wherein the modified antigen comprises an antigen fused to a targeting peptide.

[0210] Embodiment 64 The method of embodiment 60 or 61, wherein the modified antigen comprises an antigen fused to a lipid.

[0211] Embodiment 65 The method of embodiment 60 or 61, wherein the modified antigen comprises an antigen fused to a carbohydrate.

[0212] Embodiment 66. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is an antigen associated with the transplant tissue.

[0213] Embodiment 67. The method of any one of embodiments 1, 4, 5, 8-10 and 13-50, wherein the antigen is associated with a virus.

[0214] Embodiment 68. The method of any one of embodiments 1, 4, 10, 13 and 16 to 67, wherein the cell suspension is contacted with an antigen prior to, simultaneously with, or after passing through the constriction.

[0215] Embodiment 69. The method of any one of embodiments 2-4, 7, 11-13, and 22-68, wherein the tolerogenic factor comprises a polypeptide.

[0216] Embodiment 70. The method of embodiment 69, wherein the polypeptide is IL-4, IL-10, IL-13, IL-35, IFNα or TGFβ.

[0217] Embodiment 71 The method of embodiment 69, wherein the polypeptide is a therapeutic polypeptide.

[0218] Embodiment 72. The method of embodiment 69, wherein the polypeptide is a fragment of a therapeutic polypeptide.

[0219] Embodiment 73 The method of embodiment 69, wherein the polypeptide is a therapeutic peptide.

[0220] Embodiment 74 The method of embodiment 69, wherein the polypeptide is conjugated to a carbohydrate.

[0221] Embodiment 75. The method of any one of embodiments 2-4, 7, 11-13 and 22-74, wherein the cell suspension is contacted with the tolerogenic factor prior to, simultaneously with or after passing through the constriction.

[0222] Embodiment 76. The method of any one of embodiments 1 to 75, wherein the half-life of the anucleated cells is decreased.

[0223] Embodiment 77. The method of any one of embodiments 1 to 75, wherein the half-life of the anucleated cells is increased.

[0224] Embodiment 78. The method of any one of embodiments 1-6, 18, and 22-77, wherein the immune response is suppressed by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 90% or about 100%.

[0225] Embodiment 79. The method of any one of embodiments 1-6, 18, and 22-78, wherein the suppressed immune response comprises reduced production and / or secretion of one or more inflammatory cytokines.

[0226] Embodiment 80. The method of embodiment 79, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0227] Embodiment 81. The method of any one of embodiments 1-6, 18, and 22-78, wherein the suppressed immune response comprises increased production and / or secretion of one or more anti-inflammatory cytokines.

[0228] Embodiment 82. The method of embodiment 81, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor beta (TGFβ).

[0229] Embodiment 83. The method of any one of embodiments 1-6, 18, and 22-78, wherein the suppressed immune response comprises a decrease in T cell response.

[0230] Embodiment 84 The method of embodiment 83, wherein the reduced T cell response comprises reduced T cell activation.

[0231] Embodiment 85 The method of embodiment 83 or 84, wherein the reduced T cell response comprises reduced T cell survival.

[0232] Embodiment 86. The method of any one of embodiments 83 to 85, wherein the reduced T cell response comprises reduced T cell proliferation.

[0233] Embodiment 87. The method of any one of embodiments 83 to 86, wherein the reduction in T cell response comprises a reduction in T cell functionality.

[0234] Embodiment 88. The method of any one of embodiments 83 to 87, wherein the reduction in T cell response comprises a change in T cell phenotype.

[0235] Embodiment 89. The method of any one of embodiments 1-6, 18, and 22-88, wherein the immune response that is suppressed comprises non-costimulatory activation of T cells.

[0236] Embodiment 90. The method of any one of embodiments 1-6, 18, and 22-89, wherein the suppressed immune response comprises an enhancement of a Treg response.

[0237] Embodiment 91. The method of any one of embodiments 1-6, 18, and 22-90, wherein the suppressed immune response comprises a decrease in a B cell response.

[0238] Embodiment 92 The method of embodiment 91, wherein the reduced B cell response comprises reduced antibody production.

[0239] Embodiment 93. The method of any one of embodiments 1-6, 18, and 22-92, wherein the suppressed immune response comprises a decrease in cytokine production.

[0240] Embodiment 94. The method of any one of embodiments 1-6, 18, and 22-93, wherein the suppressed immune response comprises a reduction in an autoimmune response.

[0241] Embodiment 95. The method of any one of embodiments 1-6, 18, and 22-94, wherein the suppressed immune response comprises a reduced allergic response.

[0242] Embodiment 96. The method of any one of embodiments 1-6, 18, and 22-95, wherein the suppressed immune response comprises a decrease in the immune response to the transplanted tissue.

[0243] Embodiment 97. The method of any one of embodiments 1-6, 18, and 22-95, wherein the suppressed immune response comprises a reduction in a pathogenic immune response to the virus.

[0244] Embodiment 98. The method of any one of embodiments 1-6, 18, and 22-95, wherein the suppressed immune response comprises a decrease in the immune response to a therapeutic agent.

[0245] Embodiment 99. The method of any one of embodiments 1-6, 18, and 22-95, wherein the suppressed immune response comprises a decrease in the immune response to a therapeutic vehicle.

[0246] Embodiment 100. The method of any one of embodiments 10-15 and 19-77, wherein the tolerance comprises reduced production and / or secretion of one or more proinflammatory cytokines.

[0247] Embodiment 101. The method of embodiment 100, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0248] Embodiment 102. The method of any one of embodiments 10-15 and 19-77, wherein the tolerance comprises increased production and / or secretion of one or more anti-inflammatory cytokines.

[0249] Embodiment 103. The method of embodiment 102, wherein the one or more anti-inflammatory cytokines are selected from the group consisting of IL-4, IL-10, IL-13, IL-35, IFN-α and transforming growth factor beta (TGFβ).

[0250] Embodiment 104. The method of any one of embodiments 10-15 and 19-77, wherein the tolerance comprises a decrease in T cell responses.

[0251] Embodiment 105 The method of embodiment 104, wherein the reduced T cell response comprises reduced T cell activation.

[0252] Embodiment 106 The method of embodiment 104 or 105, wherein the reduced T cell response comprises reduced T cell survival.

[0253] Embodiment 107. The method of any one of embodiments 104 to 106, wherein the reduction in T cell response comprises a reduction in T cell proliferation.

[0254] Embodiment 108. The method of any one of embodiments 104 to 107, wherein the reduction in T cell response comprises a reduction in T cell functionality.

[0255] Embodiment 109. The method of any one of embodiments 104 to 108, wherein the reduction in T cell response comprises a change in T cell phenotype.

[0256] Embodiment 110. The method of any one of embodiments 10-15, 19-77, and 104-109, wherein the tolerance comprises non-costimulatory activation of T cells.

[0257] Embodiment 111. The method of any one of embodiments 10-15, 19-77, and 104-110, wherein the tolerance comprises an enhanced Treg response.

[0258] Embodiment 112. The method of any one of embodiments 10-15, 19-77, and 104-111, wherein the tolerance comprises a decrease in B cell responses.

[0259] Embodiment 113 The method of embodiment 112, wherein the reduced B cell response comprises reduced antibody production.

[0260] Embodiment 114. The method of any one of embodiments 10-15, 19-77, and 104-113, wherein the tolerance comprises a decrease in cytokine production.

[0261] Embodiment 115. The method of any one of embodiments 10-15, 19-77, and 104-114, wherein the tolerance comprises a reduction in an autoimmune response.

[0262] Embodiment 116. The method of any one of embodiments 10-15, 19-77, and 104-115, wherein the tolerance comprises a reduction in an allergic response.

[0263] Embodiment 117. The method of any one of embodiments 10-15, 19-77, and 104-116, wherein the tolerance comprises a decrease in the immune response to the transplanted tissue.

[0264] Embodiment 118. The method of any one of embodiments 10-15, 19-77, and 104-116, wherein the tolerance comprises a reduction in a pathogenic immune response to the virus.

[0265] Embodiment 119. The method of any one of embodiments 10-15, 19-77, and 104-116, wherein the tolerance comprises a decreased immune response to the therapeutic agent.

[0266] Embodiment 120. The method of any one of embodiments 10-15, 19-77, and 104-116, wherein the tolerance comprises a decreased immune response to the therapeutic vehicle.

[0267] Embodiment 121. The method according to any one of embodiments 1 to 120, which is repeated at least 1, 2, 3, 4, 5 or 6 times.

[0268] Embodiment 122. The method of embodiment 121, wherein the time interval between any two repetitions of the method is at least 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year.

[0269] Embodiment 123. A system comprising the constriction, a cell suspension and an antigen for use in the method according to any one of embodiments 1, 4, 10, 13 and 16 to 120.

[0270] Embodiment 124. A system comprising the constriction, a cell suspension and a tolerogenic factor for use in the method according to any one of embodiments 2 to 4, 7, 11 to 13 and 22 to 120. EXAMPLES

[0271] The following examples are presented for illustrative purposes of various embodiments of the present disclosure, and are not intended to limit the present disclosure in any manner. Those skilled in the art will readily appreciate that the present disclosure is fully adapted to carry out the objects and obtain the results and advantages described, as well as those objects, results, and advantages inherently suggested herein. Those skilled in the art will envision modifications thereof and other uses that are encompassed within the spirit of the present disclosure, as defined by the scope of the claims.

[0272] Example 1 Constriction-mediated delivery of dextran and IgG antibodies to human RBCs. Preface To assess filter-mediated delivery of molecules to anucleated cells, human RBCs mixed with fluorescent dextran particles or IgG antibodies were passed through syringe filters containing pores of defined sizes, and intracellular particle delivery was assessed by FACS analysis.

[0273] material and method Polycarbonate membrane filters with 2 μm diameter pore size with commercial filter and holder combination (COTS filters) were obtained from STERLITECH™. Human RBCs were separated from whole blood or leukoreduction collars using Ficoll gradient separation and resuspended in Optimem at the desired concentration (50-500 M / mL). Forceps were used to float the polycarbonate membrane filter in Optimem to wet the membrane filter. The plastic filter holder was removed from the lid and the filter was placed on the inner surface, shiny side up, and the filter holder was recapped. Dextran particles and IgG antibodies were suspended at 0.1 mg / mL. 1 mL of cells mixed with dextran particles or IgG antibodies was added to the filter holder at room temperature. Cells were forced through the filter by manual finger pressure of the syringe. The filter flow-through was collected in a 15 ml Falcon tube or FACS tube. All samples were centrifuged prior to FACS analysis and washed three times at 400rcf for 4 minutes at 4°C to remove extracellular dextran. The following steps were performed in preparation for FACS analysis: FACS buffer was prepared (PBS + final concentration: 1% FBS + 2mM EDTA), 400μL of FACS buffer was added per tube, and cells were resuspended. Voltages for forward scatter, side scatter, and fluorescence channels of the flow cytometer were adjusted to visualize all cellular events, and more than 10,000 events were recorded per sample. Cells mixed with dextran particles or IgG antibody but not passed through a filter were used as a control for delivery by endocytosis. These cells were contacted with the dye but not passed through a filter. All other steps were the same for the endocytosis control.

[0274] result RBCs mixed with 10 kDa Alexa Fluor® 647 dextran particles or 150 kDa IgG antibody conjugated to Alexa Fluor® 647 were passed through filter pores of 2 μm size. Exemplary flow cytometry histogram plots showing increased delivery of fluorescent dextran and IgG after constriction-mediated delivery (SQZ) versus endocytosis control are shown in FIG. 1A and FIG. 1B. Delivery efficiency as indicated by the percentage of cells positive for the dextran particles of IgG antibody after filter delivery is shown in FIG. 2A. Delivery efficiency of both molecules in the endocytosis control was less than 10%, whereas delivery efficiency of IgG antibody after filter delivery was 88.6% and dextran particles was 95.6%.

[0275] Cell viability was measured after filter delivery. Estimated cell viability, as indicated by the percentage of cells present within the FSC and SSC gates after filter delivery, is shown in Figure 2B. The estimated viability of cells delivered with IgG antibody after filter delivery was 68.8%, and the estimated viability of cells delivered with dextran particles was 63.3%.

[0276] Constriction-mediated delivery of dextran particles and IgG antibodies to human RBCs was achieved.

[0277] Example 2 Constriction-mediated delivery of dextran and IgG antibodies to mouse RBCs. Preface To assess filter-mediated delivery of molecules to anucleated cells, mouse RBCs mixed with fluorescent dextran particles or IgG antibodies were passed through syringe filters containing pores of defined sizes, and intracellular particle delivery was assessed by FACS analysis.

[0278] material and method Polycarbonate membrane filters with pore sizes of 1 μm and 2 μm diameter with commercial filter and holder combinations (COTS filters) were obtained from STERLITECH™. Whole blood was spun down and cells were resuspended in Optimem at the desired concentration (100-500 M / ml). Forceps were used to suspend the polycarbonate membrane filter in Optimem to wet the membrane filter. The lid of the plastic filter holder was removed and the filter was placed on the inner surface, shiny side up, and the filter holder was recapped. Dextran particles and IgG antibodies were suspended at 0.1 mg / mL. 1 mL of cells mixed with dextran particles or IgG antibodies was added to the filter holder at room temperature. Cells were forced through the filter by manually finger pressing the syringe. Alternatively, a controlled pressure system was used to propel 200 μL of cells mixed with dextran particles or IgG antibodies through the filter. The filter flow-through was collected in a 15 ml Falcon tube or FACS tube. All samples were centrifuged prior to FACS analysis and washed three times at 1000rfc for 10 min at 24°C to remove extracellular dextran. The following steps were performed in preparation for FACS analysis: FACS buffer was prepared (PBS + final concentration: 1% FBS + 2mM EDTA), 400 μL of FACS buffer was added per tube, and cells were resuspended. Voltages in the forward scatter, side scatter, and fluorescence channels of the flow cytometer were adjusted to visualize all cellular events, and more than 10,000 events were recorded per sample. Cells mixed with dextran particles or IgG antibodies but not passed through a filter were used as controls for delivery by endocytosis. These cells were contacted with the dye but not passed through a filter. All other steps were the same for the endocytosis control. NC (no contact) samples were not contacted with the dye but were subjected to the same steps as the experimental samples but were not passed through a filter.

[0279] result Mouse RBCs mixed with IgG antibody (150 kDa) were passed through filter pores of 1 μm and 2 μm size using manual syringe pressure. Exemplary flow cytometry histogram plots showing fluorescence after constriction-mediated delivery (SQZ) versus endocytosis (Endo), negative (NC), and no material control controls are shown in FIG. 3A. The "no material" control was not contacted with IgG antibody but was subjected to the same steps and passed through the filters as the experimental samples.

[0280] Cell viability was measured after filter delivery. Estimated cell viability, as indicated by the percentage of cells present within the FSC and SSC gates after filter delivery of IgG antibody using manual syringe pressure, is shown in Figure 3B. Filter-mediated delivery reduced estimated viability by approximately 70% versus controls, but resulted in a 4-12 fold higher delivery efficiency of IgG antibody, as shown in Figure 3C.

[0281] RBCs mixed with 70 kDa Alexa Fluor® 488 dextran particles and IgG antibody (150 kDa) conjugated to Alexa Fluor® 647 were passed through filter pores of 2 μm size using a controlled pressure system or using manual syringe pressures of 2 psi, 4 psi, 6 psi, 10 psi, 20 psi. Exemplary flow cytometry histogram plots are shown in Figures 4A-I, which show the fluorescence after constriction-mediated delivery (SQZ, Figures 4D-I) versus endocytosis (Endo, Figure 4A), negative (NC, Figure 4B), and no material control (Figure 4C). These data showed that filter-mediated delivery resulted in up to a 55% increase in the delivery of dextran and IgG compared to Endo, NC, and no material controls. Cell viability was also measured after filter delivery. Estimated cell viability, as indicated by the percentage of cells present within the FSC and SSC gates after filter delivery of dextran particles, is shown in Figure 5A. The delivery efficiency, as indicated by the percentage of cells positive for dextran particles or IgG antibody after filter delivery, is shown in Figure 5B. The geometric mean fluorescence after filter delivery of dextran particles or IgG antibody is shown in Figure 5C.

[0282] Using a controlled pressure system of 10 psi, 12 psi, 14 psi, 16 psi, or 18 psi, RBCs mixed with 70 kDa Alexa Fluor® 488 dextran particles were passed through filter pores of 2 μm size. Exemplary flow cytometry histogram plots showing fluorescence after constriction-mediated delivery (SQZ) versus endocytosis (Endo) and negative (NC) controls are shown in FIG. 6A. Cell viability was measured after filter delivery. Estimated cell viability, as indicated by the percentage of cells present within the FSC and SSC gates after filter delivery of dextran particles, is shown in FIG. 6B. Delivery efficiency, as indicated by the percentage of cells positive for dextran particles after filter delivery, is shown in FIG. 6C. Geometric mean fluorescence after filter delivery of dextran particles is shown in FIG. 6D.

[0283] Constriction-mediated delivery of dextran particles and IgG antibodies to mouse RBCs was achieved.

[0284] Example 3 Constriction-Mediated Delivery of Antigens to RBCs to Induce Tolerance. A series of experiments will be undertaken to demonstrate the induction of tolerance using model antigens and transgenic T cells.

[0285] RBCs are mixed with OVA antigen and passed through a constriction in a microfluidic channel, or through a surface containing holes. Pressure, temperature, and buffer composition are optimized to achieve delivery. OVA-bearing RBCs are then introduced into CD45.1 mice with CFSE-labeled adoptively transferred OT-I cells. Alternatively, OT-I T cells are activated ex-vivo before transfer into CD45.1 mice. Nine days later, mice are challenged with soluble OVA and LPS to allow for tolerogenic presentation and subsequent T cell deletion. Four days after challenge, spleens and draining lymph nodes are analyzed for OT-I proliferation.

[0286] Example 4 Constriction-Mediated Delivery of Antigens to RBCs to Induce Tolerance. To determine the ability of RBCs containing antigen delivered by SQZ to induce antigen-dependent tolerance in vivo, the numbers of antigen-specific T cells and the levels of the inflammatory cytokine IFN-γ were measured by flow cytometry.

[0287] material and method 1M OVA-specific OT-I T cells per mouse were adoptively transferred from female OT-I donor mice (CD45.2) to C57BL / 6J recipient mice (CD45.1) on day 0. On days 1 (prime) and 6 (boost), RBCs were collected from C57BL / 6J mice and either RBCs were incubated with full-length OVA protein or OVA was delivered by filter-mediated SQZ conditions, after which recipient mice were injected with 20–125M RBCs / mouse. Control animals received no injection (naive) or were injected with either PBS (challenge control) or free OVA (10 μg / animal, tolerance control). Compared to naive (no antigen or LPS), challenged animals received an antigen challenge on day 15 with an intradermal injection of 10 μg OVA protein + 50 ng LPS / mouse. OT-I T cell responses to antigen challenge were measured by flow cytometry for OT-I T cell numbers and IFN-γ intracellular staining on day 19. A schematic of the treatment schedule is shown in Figure 7 and treatment groups are summarized in Table 1. [Table 1]

[0288] result OT-I specific T cell numbers were measured in mice challenged with antigen (OVA) and adjuvant (LPS) for groups treated with either RBCs incubated with OVA protein (RBC-Endo) or RBCs with OVA protein delivered intracellularly by SZQ (RBC-SQZ) (Figure 8). These conditions were compared to animals that did not receive antigen, adjuvant, or RBCs (naive), or to mice treated with free OVA protein (tolerance control) or PBS (challenge control) followed by antigen + adjuvant challenge. OT-I T cell numbers in both draining lymph nodes and spleens were significantly inhibited in mice primed / boosted with RBCs SQZed with OVA protein compared to challenge controls ( ****P<0.0001). These mice had similar numbers of OT-I T cells as naïve mice or tolerant controls, whereas treatment with RBCs incubated with OVA protein was not different from challenge controls.

[0289] To assess the functional effects of SQZ RBC-mediated tolerance on T cell function, the % of splenic T cells that expressed high levels of IFN-γ among OT-I T cells restimulated with SIINFEKL peptide (SEQ ID NO: 1) (active OVA epitope) (Figure 9 left) and the level of IFN-γ production per cell (Figure 9 right; based on total CD44hi cells) were assessed for all groups by staining for intracellular cytokines. OT-I T cells from mice treated with OVA-SQZed RBCs showed a moderate but significant reduction in high IFN-γ expressing cells ( ** P < 0.005) and a large decrease in IFN-γ production ( **** P < 0.0001) and tolerant controls (free OVA; ** P<0.005). However, RBCs incubated with OVA protein did not show a decrease in IFN-γ production compared to challenge controls.

[0290] Representative flow cytograms for the percentage of OT-I T cells among total CD8+ T cells and the percentage of IFN-γ-responsive T cells among OT-I T cells (FIG. 10, summarized in Table 2) show that RBC-SQZ samples had less OT-I and lower IFNg responses compared to challenge control and RBC-Endo conditions. [Table 2]

[0291] Example 5 CD4+ / CD8+-mediated tolerance To determine the ability of red blood cells (RBCs) containing antigens delivered by SQZ to induce CD4- and / or CD8-mediated T cell antigen-dependent tolerance in vivo, the number of antigen-specific T cells and the levels of the inflammatory cytokine IFN-γ are measured by flow cytometry.

[0292] material and method On day 0, 1M OVA-specific CD8+ OT-I T cells and 2-4M CD4+ OT-II T cells (both CD45.2+) per mouse are adoptively transferred from female donor mice into C57BL / 6J recipient mice (CD45.1+). On days 1 (prime) and 6 (boost), RBCs are collected from C57BL / 6J mice and either RBCs are incubated with full-length OVA protein or OVA is delivered by SQZ conditions, after which recipient mice are injected with 20-125M RBCs / mouse. Control animals receive injections of either PBS (challenge control) or free OVA (10 μg / animal, tolerance control). Antigen challenge is performed on day 15 in challenge animals with an intradermal injection of 10 μg OVA protein + 50 ng LPS / mouse. On day 19, the draining lymph nodes and spleens of each mouse are analyzed and OT-I and OT-II T cell responses to antigen challenge are measured by flow cytometry for OT-I and OT-II T cell numbers and IFN-γ intracellular staining, and by ELISpot assays. A schematic of a representative treatment schedule is shown in Figure 7 and treatment groups are summarized in Table 3. [Table 3]

[0293] Example 6A Prevention in a mouse type I diabetes model Preface To determine the ability of RBCs containing antigen delivered by SQZ to induce antigen-dependent tolerance in a preventive in vivo model of murine type I diabetes, blood glucose levels are measured weekly over time following tolerance induction.

[0294] material and method Treat NOD / ShiltJ mice with RBCs at 10 weeks of age. On days 0 (prime) and 5 (boost), collect RBCs from NOD / ShiltJ mice and inject the RBCs with insulin B chain peptide 9-23 (InsB 9~23 :CKKGSSHLVEALYLVCGERG, SEQ ID NO: 2) or InsB 9~23 After delivery via SQZ conditions, recipient mice are injected with 20-125M RBCs / mouse. Control animals are injected with PBS (challenge control). Beginning 7 days after treatment (11 weeks of age), blood glucose measurements are performed weekly. Mice are considered diabetic when blood glucose levels exceed 260 mg / dL as measured by Bayer Contour Diabetes Meter / Glucose Test Strips. A schematic of a representative treatment schedule is shown in Figure 11A, and treatment groups are summarized in Table 4. [Table 4]

[0295] Example 6B Treatment of mouse type I diabetes models Preface To determine the ability of RBCs containing antigen delivered by SQZ to induce antigen-dependent tolerance in an in vivo model for the treatment of mouse type I diabetes, blood glucose levels are measured weekly over time following tolerance induction in the mice.

[0296] material and method To induce rapid onset of T1D in recipients, CD4 T cells are harvested from BDC2.5 NOD mice, activated ex vivo with p31 mimetope peptide (YVRPLWVRME, SEQ ID NO: 3) for 4 days, and adoptively transferred into normoglycemic NOD recipients (5M cells / mouse). Tolerance induction begins 8 hours after adoptive transfer and is repeated every 3 days for a total of 3 doses. Recipients are treated with PBS (challenge control) or 20-125M RBCs incubated with p31 (RBC-Endo) or 20-125M RBCs delivered by SQZ with p31 (RBC-SQZ). Blood glucose is measured daily by Bayer Contour Diabetes Meter / Glucose Test Strips. Mice are considered diabetic when blood glucose levels exceed 260 mg / dL. A schematic of a representative treatment schedule is shown in FIG. 11B, and treatment groups are summarized in Table 5. [Table 5]

[0297] Example 7A Prevention of MS-type autoimmune disorders in mice Preface To determine the ability of antigen-containing RBCs delivered by SQZ to induce antigen-dependent tolerance in an in vivo prophylactic model of murine MS-type autoimmune disorder, clinical scores of motility will be assessed daily over time.

[0298] material and method Treat female C57BL / 6 mice (10-12 weeks old) with RBCs prior to induction of experimental autoimmune encephalomyelitis (EAE). Collect RBCs from C57BL / 6 mice on day -7 (prime) and day -2 (boost) and inoculate the RBCs with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG) without stenosis. 35~55 :MEVGWYRSPFSRVVHLYRNGKGS, SEQ ID NO: 4) or OVA 323~339Incubated with peptide (negative control: ISQAVHAAHAEINEAGRGS, SEQ ID NO: 5) or MOG 35~55 After peptide delivery to RBCs by SQZ conditions, recipient mice are injected with 20–125 M RBCs / mouse. On day 0, MOG in CFA 35~55 and pertussis toxin (Hooke kit) in PBS. Mice are scored daily starting on day 7. Clinical scores are defined as follows: 1. limp tail; 2. partial hind limb paralysis; 3. complete hind limb paralysis; 4. complete hind limb and partial forelimb paralysis; and 5. moribund. A schematic of a representative treatment schedule is shown in Figure 12A and treatment groups are summarized in Table 6. [Table 6]

[0299] Example 7B Treatment of MS-type autoimmune disorders in mice Preface To determine the ability of antigen-containing RBCs delivered by SQZ to induce antigen-dependent tolerance in an established in vivo model of murine MS-type autoimmune disorder, clinical scores of motility will be assessed daily over time.

[0300] material and method Female C57BL / 6 mice (10–12 weeks old) were cultured in CFA for 1 h on day 0. 35~55 Experimental autoimmune encephalomyelitis (EAE) is induced by administration of 100 mM EDTA and pertussis toxin (Hooke kit) in PBS. Mice are then treated with RBCs on the day of EAE onset, which is the day the mice score ≥1 based on the motility criteria described below. RBCs are collected from C57BL / 6 mice on approximately days 11 / 12 (prime) and 17 / 18 (boost) and RBCs are transfected with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG) without stenosis. 35~55 :MEVGWYRSPFSRVVHLYRNGKGS, SEQ ID NO: 4) or OVA 323~339Peptide (negative control: ISQAVHAAHAEINEAGRGS, SEQ ID NO: 5) or MOG 35~55 After peptides are delivered to RBCs by SQZ conditions, recipient mice are injected with 20-125M RBCs / mouse. Mice are scored daily starting on day 19. Clinical scores are defined as follows: 1. limp tail; 2. partial hind limb paralysis; 3. complete hind limb paralysis; 4. complete hind limb and partial forelimb paralysis; and 5. moribundity. A schematic of a representative treatment schedule is shown in Figure 12B, and treatment groups are summarized in Table 7. [Table 7] [Table 8]

Claims

1. 1. A composition comprising a modified anuclear cell comprising an antigen, or an antigen and a tolerogenic factor, for suppressing an immune response or inducing tolerance in an individual, the composition comprising: passing a cell suspension comprising anucleated cells through a constriction, the constriction deforming the anucleated cells such that the antigen, or the antigen and tolerogenic factor, contacted with the cells enters the anucleated cells, thereby causing perturbation of the anucleated cells, thereby generating modified anucleated cells comprising the antigen, or the antigen and tolerogenic factor. wherein the antigen is processed in a tolerogenic environment, presentation of the antigen in the tolerogenic environment suppresses an immune response to the antigen or induces antigen-specific tolerance, the tolerogenic factor contributes to a tolerogenic environment that suppresses an immune response or contributes to antigen-specific tolerance, the anucleated cells are red blood cells, and the tolerogenic factor is (i) an immunosuppressive molecule selected from arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), nitric oxide synthase 2 (NOS2), thymic stromal lymphopoietin (TSLP), vasoactive intestinal peptide (VIP), hepatocyte growth factor (HGF), transforming growth factor beta (TGFβ), IFNα, IL-4, IL-10, IL-13, and IL-35; (ii) encodes a suppressor of a costimulatory molecule; (iii) encoding an enhancer of a coinhibitory molecule; (iv) encodes a suppressor of an inflammatory transcription factor; (v) encoding an enhancer of an anti-inflammatory transcription factor; or (vi) A composition which is any combination of (i) to (v).

2. The modified anucleated cell is (a) comprising said antigen and at least one further antigen; and / or (b) comprising said tolerogenic factor and at least one further tolerogenic factor; The composition of claim 1.

3. The composition of claim 1 or 2, wherein the constriction size is a function of the diameter of the anucleated cells.

4. 4. The composition of claim 1, wherein the constriction size is about 10%, about 20%, about 30%, about 40%, about 50%, about 60% or about 70% of the cell diameter.

5. The composition of any one of claims 1 to 4, wherein the cell suspension comprises a mixed population of cells.

6. The composition of claim 5 , wherein the cell suspension is whole blood.

7. The composition of any one of claims 1 to 4, wherein the cell suspension comprises a purified cell population.

8. 8. The composition of claim 7, wherein the cell suspension comprises a purified non-nucleated cell population, the purified non-nucleated cell population comprising red blood cells.

9. (a) the cell suspension comprises human cells; and / or (b) the anucleated cell is a human cell; The composition according to any one of claims 1 to 8.

10. The composition according to any one of claims 1 to 9, wherein the anucleated cells are derived from the individual or a different individual.

11. The antigen is (i) foreign antigen; (ii) self-antigen; (iii) allograft antigen; (iv) food allergens; (v) a viral antigen; or (vi) a therapeutic agent, the therapeutic agent comprising: (a) Clotting factors; (b) Antibodies; (c) growth factors; (d) a hormone; or (e) Recombinant cytokines a therapeutic agent, The composition according to any one of claims 1 to 10, wherein the compound is one or more of:

12. The composition of any one of claims 1 to 11, wherein the antigen is a viral antigen, the viral antigen being derived from one or more of an adenovirus, an adeno-associated virus, a baculovirus, a herpes virus or a retrovirus.

13. The composition of claim 12 , wherein the viral antigen is derived from an adeno-associated virus.

14. The antigen is a therapeutic agent, the therapeutic agent comprising: (a) Clotting factors; (b) Antibodies; (c) growth factors; (d) a hormone; or (e) Recombinant cytokines The composition according to any one of claims 1 to 12, wherein the compound is one or more of:

15. The antigen is (i) a protein or polypeptide; (ii) a lipid antigen; or (iii) carbohydrate antigens The composition according to any one of claims 1 to 14,

16. The composition of any one of claims 1 to 11, wherein the antigen is present in a cell lysate.

17. The composition of any one of claims 1 to 16, wherein the antigen is a modified antigen.

18. The modified antigen is (a) an antigen fused to a polypeptide; (b) an antigen fused to a therapeutic agent; (c) an antigen fused to a targeting peptide; (d) an antigen fused to a lipid; and / or (e) Antigens fused to carbohydrates 20. The composition of claim 17, comprising:

19. The composition of any one of claims 1 to 18, wherein the tolerogenic agent comprises a polypeptide or a fragment of a polypeptide.

20. The composition of any one of claims 1 to 19, wherein the suppressed immune response or induced tolerance comprises reduced production and / or secretion of one or more pro-inflammatory cytokines.

21. The composition of claim 20, wherein the one or more inflammatory cytokines are selected from the group consisting of interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF).

22. The composition of any one of claims 1 to 19, wherein the suppressed immune response or induced tolerance comprises a reduced T cell response.

23. The reduction in the T cell response (a) decreased T cell activation; (b) decreased T cell survival; (c) decreased T cell proliferation; (d) a decrease in T cell functionality; and / or (e) Changes in T cell phenotype 23. The composition of claim 22 comprising:

24. The composition of any one of claims 1 to 23, wherein the suppressed immune response or induced tolerance comprises a reduction in an autoimmune response.

25. 25. The composition of claim 24, wherein the reduction in autoimmune response comprises a reduction in immune response to antigens associated with type I diabetes, rheumatoid arthritis, psoriasis, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Crohn's disease, or ulcerative colitis.

26. The composition of any one of claims 1 to 23, wherein the suppressed immune response or induced tolerance comprises a reduced allergic response.

27. 27. The composition of claim 26, wherein the reduced allergic response is to a food allergen.

28. The suppressed immune response or induced tolerance is (a) a reduction in the immune response to the transplanted tissue; (b) a reduction in the pathogenic immune response to the virus; (c) a decrease in the immune response to the therapeutic agent; and / or (d) a reduction in the immune response to the therapeutic vehicle The composition of any one of claims 1 to 23, comprising:

29. The suppressed immune response or induced tolerance is (a) a reduction in the immune response to a therapeutic agent, the therapeutic agent being (i) a clotting factor or (ii) a hormone; and / or 30. The composition of claim 28, wherein (b) the composition comprises a reduced immune response to a therapeutic vehicle, the therapeutic vehicle being a virus.

30. The suppressed immune response or induced tolerance comprises a decrease in the immune response to a therapeutic agent, the therapeutic agent being (i) a coagulation factor, the coagulation factor being factor VIII or factor IX; or (ii) a hormone, the hormone being insulin, human growth hormone or follicle-stimulating hormone; 30. The composition of claim 29.

31. 30. The composition of claim 28 or 29, wherein the suppressed immune response or induced tolerance comprises a decrease in the immune response to a virus, wherein the virus is one or more of an adenovirus, an adeno-associated virus, a baculovirus, a herpes virus or a retrovirus.

32. 32. The composition of claim 31 , wherein the virus is an adeno-associated virus.

33. A composition comprising a modified anuclear cell comprising an antigen, or an antigen and a tolerogenic factor, the composition comprising: (a) passing a cell suspension containing anucleated cells through a constriction that deforms the cells, the diameter of the constriction being a function of the diameter of the anucleated cells in the suspension, thereby causing a perturbation of the anucleated cells sufficient to allow the antigen, or the antigen and the tolerogenic factor, to pass through and form a perturbed anucleated cell; (b) incubating the perturbed anucleated cell with the antigen, or the antigen and the tolerogenic factor, for a time sufficient for the antigen, or the antigen and the tolerogenic factor, to enter the perturbed anucleated cell, thereby generating the modified anucleated cell comprising the antigen, or the antigen and the tolerogenic factor; wherein the anucleated cells are red blood cells, and the tolerogenic factor is (i) an immunosuppressive molecule selected from arginase-1 (ARG1), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), nitric oxide (NO), nitric oxide synthase 2 (NOS2), thymic stromal lymphopoietin (TSLP), vasoactive intestinal peptide (VIP), hepatocyte growth factor (HGF), transforming growth factor beta (TGFβ), IFNα, IL-4, IL-10, IL-13, and IL-35; (ii) encodes a suppressor of a costimulatory molecule; (iii) encoding an enhancer of a coinhibitory molecule; (iv) encodes a suppressor of an inflammatory transcription factor; (v) encoding an enhancer of an anti-inflammatory transcription factor; or (vi) A composition which is any combination of (i) to (v).

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