ALLEVIATING GRAFT VERSUS HOST DISEASE USING ENGINEERED INKT CELLS
20260034218 ยท 2026-02-05
Assignee
Inventors
Cpc classification
F03B17/00
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
A61K40/15
HUMAN NECESSITIES
F03G3/094
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
F03B17/025
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
F05B2220/602
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
F03G7/104
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
A61P37/06
HUMAN NECESSITIES
F03B17/005
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
International classification
A61K40/15
HUMAN NECESSITIES
Abstract
We have discovered that allogeneic HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells display potent anti-GvHD functions, by eliminating antigen-presenting myeloid cells in vitro and in xenograft models, without negatively impacting tumor eradication by allogeneic T cells in preclinical models of lymphoma and leukemia. The .sup.3rdHSC-iNKT cells closely resembled the CD4.sup.CD8.sup./+ subsets of endogenous human iNKT cells in phenotype and functionality. Embodiments of the invention harness these discoveries in new methods and materials for alleviating graft versus host disease.
Claims
1. A method of inhibiting or treating a graft versus host disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of allogeneic HSC-engineered human iNKT cells such that graft versus host disease is inhibited or treated in the subject.
2. The method of claim 1, wherein the graft versus host disease is selected from the group consisting of acute graft-versus-host-disease (aGVHD) and chronic graft-versus-host-disease (cGVHD).
3. The method of claim 1, wherein the subject has been diagnosed with hematologic malignancy.
4. The method of claim 1, wherein the subject has undergone or will undergo an allogeneic hematopoietic stem cell transplantation procedure.
5. The method of claim 4, wherein the subject is administered the allogeneic HSC-engineered human iNKT cells at the time the subject undergoes the allogeneic hematopoietic stem cell transplantation procedure.
6. The method of claim 5, wherein the subject is administered allogeneic HSC-engineered human iNKT cells mixed with allogeneic hematopoietic stem cells.
7. The method of claim 1, wherein the subject is administered at least 110.sup.6 allogeneic HSC-engineered human iNKT cells.
8. The method of claim 1, wherein the subject is administered at least 0.03110.sup.6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells.
9. The method of claim 1, wherein the engineered iNKT cells comprise one or more exogenous nucleic acids transduced therein, wherein: the one or more exogenous nucleic acids comprise a V24-J18 iNKT cell receptor gene; and/or the one or more exogenous nucleic acids comprise a classical or T cell receptor gene.
10. A method of depleting allogeneic CD14.sup.+ myeloid cells from a subject transfused with allogenic leukocytes including the allogeneic CD14.sup.+ myeloid cells, the method comprising administering to said subject amounts of allogeneic HSC-engineered human iNKT cells sufficient to target the allogeneic CD14.sup.+ myeloid cells in the subject, thereby depleting the HSC-engineered human iNKT cells in the subject.
11. The method of claim 10, wherein the allogeneic HSC-engineered human iNKT cells comprise one or more exogenous nucleic acids, wherein: the one or more exogenous nucleic acids comprise a V24-J18 iNKT cell receptor gene; and/or the one or more exogenous nucleic acids comprise a classical or T cell receptor gene.
12. The method of claim 10, wherein the subject is administered at least 1 10.sup.6 allogeneic HSC-engineered human iNKT cells.
13. The method of claim 10, wherein the subject is administered allogeneic HSC-engineered human iNKT cells at the time that the subject is transfused with the allogenic leukocytes.
14. The method of claim 10, wherein the subject has been diagnosed with hematologic malignancy.
15. A method of inhibiting or suppressing expansion of Th1-type pathogenic donor T cells in a subject treated with allogenic T cells in a therapeutic regimen, the method comprising administering to said subject amounts of allogeneic HSC-engineered human iNKT cells sufficient to inhibit or suppress the expansion of Th1-type pathogenic donor T cells in the subject.
16. The method of claim 15, wherein the subject is administered at least 110.sup.6 allogeneic HSC-engineered human iNKT cells.
17. The method of claim 15, wherein the subject is administered allogeneic HSC-engineered human iNKT cells at the time that the subject is treated with the allogenic T cells in the therapeutic regimen.
18. The method of claim 15, wherein the allogeneic HSC-engineered human iNKT cells: are derived from hematopoetic stem cells transduced with one or more exogenous nucleic acids comprising a V24-J18 T cell receptor gene receptor gene.
19. The method of claim 18, wherein the subject is selected to be a patient diagnosed with hematologic malignancy.
20. The method of claim 19, wherein the subject is administered at least 0.03110.sup.6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0019] In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the aspects of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. The following text discusses various embodiments of the invention.
[0020] Invariant nature killer T (iNKT) cells have been studied extensively for their roles in modulating GvHD and GvL. iNKT cells are a small subset of T cells that express both a semi-invariant T cell receptor (V24-J18 in humans and Va14-J18 in mice paired with a limited selection of VP chains) and natural killer cell markers (e.g., CD161 in humans and NK1.1 in mice) (Bendelac et al., 2007; Brennan et al., 2013; Brigl and Brenner, 2004; Kronenberg, 2005; Kumar et al., 2017; Lantz and Bendelac, 1994; Taniguchi et al., 2003). Unlike conventional TCRs that recognize peptide antigens presented on classical polymorphic MHC Class I and II molecules, the iNKT TCR recognizes glycolipid antigens presented on non-polymorphic MHC Class I-like molecule CD1d (Cohen et al., 2009). iNKT cells in mouse comprise CD4+ and CD4.sup.CD8.sup. (double negative, DN) subsets (Brigi and Brenner, 2004), and iNKT cells in human comprise CD4.sup.+, CD8.sup.+ and DN subsets (Brigl and Brenner, 2004). iNKT cells express high levels of cytokine mRNA and produce large amounts of cytokines upon primary stimulation (Brigl and Brenner, 2004). iNKT cell subset have differential cytokine patterns and cytolytic functions: CD4.sup.+ iNKT cell subset produce much higher levels of IL-4 as compared to CD8.sup.+ and DN subsets; the latter subsets express much higher levels of Granzyme B and Perforin and have stronger cytolytic function as compared to the former (Brigl and Brenner, 2004).
[0021] Here, we report a new use for allogeneic (third-party) HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells. The .sup.3rdHSC-iNKT cells of the invention closely resemble the CD4.sup.CD8.sup./+ subsets of endogenous human iNKT cells. We have further discovered that these cells display potent anti-GvHD functions and can, for example, eliminate antigen-presenting myeloid cells in vitro and in xenograft preclinical models of lymphoma and leukemia without negatively impacting tumor eradication by allogeneic T cells.
[0022] As discussed below, the invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, methods of inhibiting or treating a graft versus host disease in a subject/patient in need thereof. These methods include administering to the subject a therapeutically effective amount of allogeneic HSC-engineered human iNKT cells (e.g., at least 0.03110.sup.6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells) such that graft versus host disease is inhibited or treated in the subject. In this context, embodiments of the invention can be used to treat acute graft-versus-host-disease (aGVHD) and/or chronic graft-versus-host-disease (cGVHD). Typically in these methods, the engineered iNKT cells comprise one or more exogenous nucleic acids that has been transduced therein. In illustrative embodiments, the one or more exogenous nucleic acids comprise a V24-J18 iNKT cell receptor gene; and/or the one or more exogenous nucleic acids comprise a classical or T cell receptor gene.
[0023] In certain embodiments of the invention, the subject/patient has been diagnosed with hematologic malignancy such as a leukemia or a lymphoma. In some embodiments of the invention, the subject is selected to be patient who has undergone or will undergo an allogeneic hematopoietic stem cell transplantation procedure. In certain embodiments, the subject is administered the allogeneic HSC-engineered human iNKT cells at the time the subject undergoes the allogeneic hematopoietic stem cell transplantation procedure. In some embodiments of the invention, the subject is administered allogeneic HSC-engineered human iNKT cells mixed or in combination with allogeneic hematopoietic stem cells. In some embodiments, the subject is administered at least 110.sup.6 allogeneic HSC-engineered human iNKT cells. In certain embodiments, the subject is administered at least 0.03110.sup.6 cells/kg of body weight of the allogeneic HSC-engineered human iNKT cells.
[0024] Embodiments of the invention also include methods of depleting allogeneic CD14.sup.+ myeloid cells from a subject transfused with allogenic leukocytes (including the allogeneic CD14.sup.+ myeloid cells). See, for example, the data presented in
[0025] Embodiments of the invention also include methods of inhibiting or suppressing expansion of Th1-type pathogenic donor T cells in a subject (e.g., one selected to be a patient diagnosed with hematologic malignancy) treated with allogenic T cells in a therapeutic regimen. See for example the discussion below and
[0026] In certain embodiments, HSC-engineered human iNKT cells are lacking or have reduced surface expression of at least one HLA-I or HLA-II molecule. In some embodiments, a lack of surface expression of HLA-I and/or HLA-II molecules is achieved by disrupting the genes encoding individual HLA-I/II molecules, or by disrupting the gene encoding B2M (beta 2 microglobulin) that is a common component of all HLA-I complex molecules, or by disrupting the genes encoding CIITA (the class II major histocompatibility complex transactivator) that is a critical transcription factor controlling the expression of all HLA-IL genes. See, e.g., WO 2019/241400, the contents of which are incorporated herein by reference.
[0027] Related embodiments of the invention also include, for example, methods of inhibiting or treating a graft versus host disease in a patient, by administering to the patient a therapeutically effective amount of the third-party HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells disclosed herein. In certain embodiments of the invention, the patient has been diagnosed with hematologic malignancy. In typical embodiments of the invention, the patient has undergone or will undergo an allogeneic hematopoietic stem cell transplantation procedure as part of a treatment regimen for the hematologic malignancy. In certain embodiments, the patient is administered the third-party HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells at the time the subject undergoes the allogeneic hematopoietic stem cell transplantation procedure. Optionally the patient is administered third-party HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells mixed with allogeneic hematopoietic stem cells.
[0028] In illustrative embodiments of the invention, the patient is administered at least 110.sup.6 third-party HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells. In certain embodiments, the patient is administered at least 0.03110.sup.6 cells/kg of body weight of the third-party HSC-engineered human iNKT (.sup.3rdHSC-iNKT) cells. In some embodiments of the invention, the engineered iNKT cells comprise one or more exogenous nucleic acids transduced therein. Optionally, for example, the one or more exogenous nucleic acids comprise a T cell receptor alpha chain gene and/or a T cell receptor alpha chain gene; and the engineered iNKT cells comprise clonal populations of cells comprising the T cell receptor alpha chain gene and/or the T cell receptor beta chain gene.
[0029] In the present disclosure, a patient/subject can be someone diagnosed with a disease or disorder. The disease or disorder may be at least one of a hemoglobinopathy, a congenital hemoglobinopathy, -Thalessemia major, sickle cell disease (SCD), severe aplastic anemia, Fanconi's anemia, dyskeratosis congenita, Blackfan-Diamond anemia, Thalassemia, congenital amegakaryocytic thrombocytopenia, severe combined immunodeficiency, T cell immunodeficiency, T cell immunodeficiency-SCID variants, Wiskott-Aldrich syndrome, a hemophagocytic disorder, a lymphoproliferative disorder, severe congenital neutropenia, chronic granulomatous disease, a phagocytic cell disorder, IPEX syndrome, juvenile rheumatoid arthritis, systemic sclerosis, an autoimmune disorder, an immune dysregulation disorder, mucopolysaccharoidoses, MPS-I, MPS-VI, osteopetrosis, a metabolic disease, globoid cell leukodystrophy (Krabbe), metachromatic leukodystrophy, cerebral X-linked adrenoleukodystrophy, a myelofibrosis disease, a myeloproliferative disease, a plasma cell disorder, a mast cell disease, common variable immunodeficiency, chronic granulomatous disease, multiple sclerosis, systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and polymyositis-dermatomyositis. For example, the subject can be diagnosed with a disease or disorder and be designated to undergo an allogeneic transplant. Alternatively, the subject can be diagnosed with a disease or disorder and has previously undergone an allogeneic transplant.
[0030] In the present disclosure the subject can be diagnosed with a cancer. The cancer may be a hematological cancer. The cancer can be at least one of Acute myeloid leukemia, myelodysplastic syndrome, follicular lymphoma, diffuse large B cell lymphoma, acute lymphoblastic leukemia, multiple myeloma, Hodgkin lymphoma, chronic myeloid leukemia, T cell non-Hodgkin lymphoma, lymphoblastic B cell non-Hodgkin lymphoma (non-Burkitt), Burkitt's lymphoma, anaplastic large cell lymphoma, germ cell tumor, Ewing's sarcoma, soft tissue sarcoma, neuroblastoma, Wilms' tumor, osteosarcoma, medulloblastoma, acute promyelocytic leukemia, mantle cell lymphoma, T cell lymphoma, lymphoplasmacytic lymphoma, cutaneous T cell lymphoma, plasmablastic lymphoma, chronic lymphocytic leukemia, breast cancer and renal cancer. For example, the subject can be diagnosed with a cancer and be designated to undergo an allogeneic transplant. Alternatively, the subject can be diagnosed with a cancer and has previously undergone an allogeneic transplant.
[0031] In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a cancer and be designated to undergo a transplant in order to treat the cancer. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a cancer and has previously undergone a transplant in order to treat the cancer.
[0032] In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a cancer and be designated to undergo an allogeneic transplant in order to treat the cancer. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a cancer and has previously undergone an allogeneic transplant in order to treat the cancer.
[0033] In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and be designated to undergo a transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and has previously undergone a transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and be designated to undergo an allogeneic transplant in order to treat the disease or disorder. In some embodiments of the methods and uses of the present disclosure, a subject can be diagnosed with a disease or disorder and has previously undergone an allogeneic transplant in order to treat the disease or disorder.
[0034] In the present disclosure, a subject can have been previously administered a transplant. Accordingly, .sup.3rdHSC-iNKT cells can be administered to the subject after the latter has been administered a transplant. In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered an allogeneic transplant. Accordingly, in some embodiments an at least one therapeutically effective amount of .sup.3rdHSC-iNKT cells can be administered to the subject after the subject has been administered an allogeneic transplant.
[0035] In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered a conditioning therapy in connection with a transplant. In some embodiments of the methods and uses of the present disclosure, a subject can have been previously administered a conditioning therapy in connection with an allogeneic transplant. In some embodiments a conditioning therapy can comprise the administration of radiation therapy, chemotherapy, radiomimetic therapy or any combination thereof. In some embodiments a radiation therapy can comprise total body irradiation.
[0036] A conditioning therapy may be administered in connection with the allogenic transplant. A conditioning therapy can comprise, such as consist of, the administration of radiation therapy, chemotherapy, radiomimetic therapy or any combination thereof. The radiation therapy can comprise total body irradiation.
[0037] The sections below disclose and describe further aspects, methods and/or materials in connection with the invention disclosed herein. Certain aspects of the invention such as supplemental materials are discussed in Li et al., iScience volume 25, issue 9, 104859, Sep. 16, 2022 (hereinafter Li et al.) the contents of which are incorporated by reference.
Illustrative Materials and Methods of the Invention
[0038] The beneficial roles of iNKT cells in reducing GvHD while retaining GvL in murine allo-HSCT has been well reported (Lan et al., 2003; Pillai et al., 2007; Schneidawind et al., 2014; Zeng et al., 1999). Previous studies demonstrated that total lymphoid irradiation (TLI) conditioning prior to allo-HSCT prevented GvHD and preserved GvL effect due to the selective depletion of host conventional T cells and relative expansion of NKT cells (Lan et al., 2003; Zeng et al., 1999). The host iNKT cells interact with donor myeloid cells to augment donor Treg expansion (Pillai et al., 2007). Addition of recipient, donor, or third-party (heterologous) iNKT cells into the allograft was also shown to significantly reduce the risk of GvHD in allo-HSCT without diminishing GvL by polarizing donor T cells to a Th2 phenotype (Schneidawind et al., 2015, 2014).
[0039] The protective roles of human iNKT cells against GvHD have also been highlighted by multiple clinical studies. Non-myeloablative conditioning with TLI/anti-Thymocyte Globulin (ATG) prior to allo-HSCT coincided with a higher iNKT/T cell ratio, decreased incidences of GvHD, and retained GvL effect (Kohrt et al., 2009; Lowsky et al., 2005). Patients with GvHD early after transplantation were found to have reduced numbers of total circulating iNKT cells (Haraguchi et al., 2004), whereas enhanced iNKT cell reconstitution following allo-HSCT positively correlated with a reduction in GvHD without loss of GvL effect (Rubio et al., 2012). In particular, high CD4.sup., but not CD4.sup.+, iNKT cell numbers in donor allograft was associated with clinically significant reduction in GvHD in patients receiving allo-HSCT (Chaidos et al., 2012). Thus, increasing the numbers of iNKT cells, particularly the CD4.sup. iNKT cells, in the allograft may provide an attractive strategy for suppressing GvHD while preserving GvL effect. Due to their recognition of non-polymorphic CD1d (Bae et al., 2019), iNKT cells can be sourced from third-party donors.
[0040] However, human periphery blood contains extremely low number and high variability of iNKT cells (0.001-1% in blood), making it challenging to expand sufficient numbers of iNKT cells for therapeutic applications (Krijgsman et al., 2018). To overcome this critical limitation, we have previously established a method to generate large amounts of human iNKT cells through TCR gene engineering of hematopoietic stem cells (HSCs) followed by in vivo reconstitution; using this method, we have successfully generated both mouse and human HSC-engineered iNKT (HSC-iNKT) cells (Li et al., 2021b; Y. R. Li et al., 2022; Smith et al., 2015; Zhu et al., 2019). While such an in vivo approach to providing iNKT cells maybe suitable for autologous transplantation, applying this for allogeneic transplantation faces significant hurdles (Smith et al., 2015; Zhu et al., 2019). Here, we intended to build on the HSC-iNKT engineering approach and develop an ex vivo culture method to produce large amounts of third party human iNKT cells; these cells can potentially be used as a universal and off-the-shelf reagent for improving allo-HSCT outcomes by ameliorating GvHD while preserving GvL effect.
Results
Ex Vivo Generation and Characterization of Human HSC-Engineered iNKT (HSC-iNKT) Cells
[0041] Cord blood (CB)-derived human CD34.sup.+ hematopoietic stem and progenitor cells (denoted as HSCs) were collected and then transduced with a Lenti/iNKT-sr39TK lentiviral vector that encodes three transgenes: a pair of iNKT TCR and chain genes as well as an sr39TK suicide/imaging report gene (
[0042] This manufacturing process of generating HSC-iNKT cells was robust and of high yield and high purity for all 9 donors tested (4 for ATO culture and 5 for Feeder-Free culture) (
[0043] To increase the safety profile of HSC-iNKT cell product, we included an sr39TK PET imaging/suicide gene in the lentiviral vector, which allows for the in vivo monitoring of these cells using PET imaging and the elimination of these cells through ganciclovir (GCV)-induced depletion in case of an adverse event (
[0044] We next studied the phenotype and functionality of HSC-iNKT cells, in comparison with healthy donor periphery blood mononuclear (PBMC)-derived iNKT (PBMC-iNKT) cells and conventional T (PBMC-Tcon). HSC-iNKT cells displayed a phenotype closely resembling PBMC-iNKT cells and distinct from PBMC-Tcon cells: they expressed high levels of memory T cell markers (i.e., CD45RO) and NK cell markers (i.e., CD161, NKG2D, and DNAM-1) and expressed exceedingly high levels of Th1 cytokines (i.e., IFN-, TNF-, and IL-2) as well as high levels of cytotoxic molecules (i.e., Perforin and Granzyme B) (
Third Party HSC-iNKT (.sup.3rdHSC-iNKT) Cells Ameliorate Xeno-GvHD in NSG Mice Engrafted with Human PBMC
[0045] The engineered HSC-iNKT cells were predominantly CD4.sup. (
[0046] To further characterize the changes in GvHD severity, the acute and chronic GvHD overlapping target organs (i.e., lung, liver and skin) and chronic GvHD prototypical target organs (i.e., salivary glands) were collected for pathological analysis on day 40 after engrafting donor PBMCs alone or together with .sup.3rdHSC-iNKT cells (Wu et al., 2013). Compared with control NSG mice, the recipient mice engrafted with PBMCs alone showed severe infiltration and damage in the liver and lung. Although the skin tissue did not have severe infiltration, there was an enlarged epidermis, a sign of excessive collagen deposition (Wu et al., 2013). The salivary gland also showed infiltration and damage of gland follicles (
[0047] Flow cytometry analysis also revealed significantly less numbers of donor T cells in the blood and spleen, as well as less T cell infiltration in GvHD target organs (i.e., lung, liver and bone marrow;
.sup.3rdHSC-iNKT cells eliminate donor CD14.sup.+ myeloid cells in part through CD1d recognition
[0048] Donor myeloid cell-derived antigen presenting cells have been reported to exacerbate acute and chronic GvHD induced by donor T cells (Anderson et al., 2005; Chakraverty and Sykes, 2007; Jardine et al., 2020). Donor T cell production of GM-CSF has also been reported to recruit donor myeloid cells, which in turn amplifies the activation of allogeneic T cells and exacerbates GvHD severity (Piper et al., 2020; Tugues et al., 2018). Consistently, we observed that removal of CD14.sup.+ myeloid cells in the PBMCs reduced xeno-GvHD in NSG recipient mice (
[0049] iNKT cells have been shown to target myeloid (i.e., tumor-associated 10 macrophages) and myelomonocytic cells (Cortesi et al., 2018; Gorini et al., 2017; Janakiram et al., 2017; Y.-R. Li et al., 2022; Song et al., 2009). To validate that the .sup.3rdHSC-iNKT cells ameliorate GvHD via depleting donor CD14.sup.+ myeloid cells in the xeno-GvHD model, we conducted another experiment wherein NSG mice received CD14.sup.+ myeloid cell-depleted PBMCs with or without the addition of .sup.3rdHSC-iNKT cells (
[0050] To study the molecular regulation of .sup.3rdHSC-iNKT cell deletion of donor CD14.sup.+ myeloid cells, we performed an in vitro mixed lymphocyte reaction (MLR) assay (
.sup.3rdHSC-iNKT Cells Preserved GvL Activity while Ameliorating GvHD
[0051] Next, we studied the potential of .sup.3rdHSC-iNKT cells to preserve graft-versus-leukemia (GvL) while ameliorate GvHD, using a human Raji B cell lymphoma and a human HL60 acute myeloid leukemia (AML) xenograft NSG mouse models. We engineered Raji and HL60 tumor cells to overexpress the firefly luciferase and EGFP dual-reporters (denoted as Raji-FG and HL60-FG, respectively) to enable the convenient measurement of tumor killing using in vitro luminescence reading or in vivo bioluminescence imaging (BLI). When co-cultured in vitro, .sup.3rdHSC-iNKT cells effectively killed the Raji-FG and HL60-FG cells via a NK activating receptor (i.e., NKG2D and DNAM-1)-mediated tumor targeting mechanism (
[0052] NSG mice were inoculated intravenously (i.v.) with Raji-FG cells, followed by adoptive transfer of healthy donor PBMCs without or with the addition of .sup.3rdHSC-iNKT cells (
Experimental Model and Subject Details
[0053] Mice
[0054] NOD.Math.Cg-Prkdc.sup.SCIDII2rg.sup.tmlwjl/SzJ (NOD/SCID/IL-2R.sup./, NSG) mice were maintained in the animal facilities at the University of California, Los Angeles (UCLA). Six- to ten-week-old mice were used for all experiments unless otherwise indicated. All animal experiments were approved by the Institutional Animal Care and Use Committee of UCLA.
Cell Lines and Viral Vectors
[0055] The murine bone marrow derived stromal cell line MS5-DLL4 was obtained from Dr. Gay Crooks' lab (UCLA). Human Raji B cell lymphoma cell line, HL60 acute myeloid leukemia cell line, and HEK 293T cell line were purchased from the American Type Culture Collection (ATCC).
[0056] Lentiviral vectors used in this study were all constructed from a parental lentivector pMNDW (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019). The Lenti/iNKT-sr39TK vector was constructed by inserting into pMNDW vector a synthetic tricistronic gene encoding human iNKT TCR-F2A-TCR-P2A-sr39TK; the Lenti/FG vector was constructed by inserting into pMNDW a synthetic bicistronic gene encoding Fluc-P2A-EGFP. The synthetic gene fragments were obtained from GenScript and IDT. Lentiviruses were produced using HEK 293T cells, following a standard calcium precipitation protocol and an ultracentrifigation concentration protocol (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019). Lentivector titers were measured by transducing HT29 cells with serial dilutions and performing digital qPCR (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
[0057] To make stable tumor cell lines overexpressing firefly luciferase and enhanced green fluorescence protein (FG) dual-reporters, parental tumor cell lines were transduced with lentiviral vectors encoding the intended gene(s). 72 h following lentiviral transduction, cells were subjected to flow cytometry sorting to isolate gene-engineered cells for making stable cell lines. Two stable tumor cell lines were generated for this study, including Raji-FG and HL60-FG.
Human Periphery Blood Mononuclear Cells (PBMCs)
[0058] Healthy donor human PBMCs were obtained from the UCLA/CFAR Virology Core Laboratory, with identification information removed under federal and state regulations. Cells were cryopreserved in Cryostor CS10 (BioLife Solution) using CoolCell (BioCision) and were stored in liquid nitrogen for all experiments and long-term storage.
Media and Reagents
[0059] -Galactosylceramide (GC, KRN7000) was purchased from Avanti Polar Lipids. Recombinant human IL-2, IL-3, IL-4, IL-7, IL-15, Flt3-Ligand, Stem Cell Factor (SCF), Thrombopoietin (TPO), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) were purchased from Peprotech. Ganciclovir (GCV) was purchased from Sigma.
[0060] X-VIVO 15 Serum-Free Hematopoietic Cell Medium was purchased from Lonza. RPMI 1640 and DMEM cell culture medium were purchased from Coming Cellgro. Fetal bovine serum (FBS) was purchased from Sigma. Medium supplements, including Penicillin-Streptomycine-Glutamine (P/S/G), MEM non-essential amino acids (NEAA), HEPES Buffer Solution, and Sodium Pyruvate, were purchased from GIBCO. Beta-Mercaptoethanol (R-ME) was purchased from Sigma. Normocin was purchased from InvivoGen. Complete lymphocyte culture medium (denoted as C10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol), P/S/G (1% vol/vol), MEM NEAA (1% vol/vol), HEPES (10 mM), Sodium Pyruvate (1 mM), (3-ME (50 mM), and Normocin (100 mg/ml). Medium for culturing human Raji and HL60 tumor cell lines (denoted as R10 medium) was made of RPMI 1640 supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol). Medium for culturing HEK 293T cell line (denoted as D10 medium) was made of DMEM supplemented with FBS (10% vol/vol) and P/S/G (1% vol/vol).
Method Details
Antibodies and Flow Cytometry
[0061] All flow cytometry stains were performed in PBS for 15 min at 4 C. The samples were stained with Fixable Viability Dye eFluor506 (e506) mixed with Mouse Fc Block (anti-mouse CD16/32) or Human Fc Receptor Blocking Solution (TrueStain FcX) prior to antibody staining. Antibody staining was performed at a dilution according to the manufacturer's instructions. Fluorochrome-conjugated antibodies specific for human CD45 (Clone H130), TCR (Clone I26), CD4 (Clone OKT4), CD8 (Clone SKI), CD45RO (Clone UCHL1), CD161 (Clone HP-3G10), CD69 (Clone FN50), CD56 (Clone HCD56), CD62L (Clone DREG-56), CD14 (Clone HCD14), CD1d (Clone 51.1), NKG2D (Clone 1D11), DNAM-1 (Clone 11A8), IFN- (Clone B27), Granzyme B (Clone QA16A02), Perforin (Clone dG9), TNF- (Clone Mab11), IL-2 (Clone MQ1-17H12), HLA-A2 (Clone BB7.2) were purchased from BioLegend; Fluorochrome-conjugated antibodies specific for human CD34 (Clone 581) and TCR V24-J18 (Clone 6B11) were purchased from BD Biosciences. Human Fc Receptor Blocking Solution (TrueStain FcX) was purchased from Biolegend, and Mouse Fc Block (anti-mouse CD16/32) was purchased from BD Biosciences. Fixable Viability Dye e506 were purchased from Affymetrix eBioscience. Intracellular cytokines were stained using a Cell Fixation/Permeabilization Kit (BD Biosciences). Stained cells were analyzed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotech). FlowJo software was utilized to analyze the data.
Enzyme-Linked Immunosorbent Cytokine Assays (ELISAs)
[0062] The ELISAs for detecting human cytokines were performed following a standard protocol from BD Biosciences. Supernatants from co-culture assays were collected and assayed to quantify IFN-. Capture and biotinylated pairs for detecting cytokines were purchased from BD Biosciences. The streptavidin-HRP conjugate was purchased from Invitrogen. Human cytokine standards were purchased from eBioscience. Tetramethylbenzidine (TMB) substrate was purchased from KPL. The samples were analyzed for absorbance at 450 nm using an Infinite M1000 microplate reader (Tecan).
In Vitro Generation of HSC-Engineered iNKT (HSC-iNKT) Cells
[0063] Cord blood-derived human CD34.sup.+ hematopoietic stem and progenitor cells (denoted as HSCs) were obtained from HemaCare. Frozen-thawed HSCs were revived in HSC-culture medium comprised of X-VIVO 15 Serum-Free Hematopoietic Cell Medium supplemented with human recombinant SCF (50 ng/ml), FLT3-L (50 ng/ml), TPO (50 ng/ml), and IL-3 (10 ng/ml) for 24 hours. Cells were then transduced with Lenti/iNKT-sr39TK viruses for another 24 hours (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019). The transduced HSCs were then collected and put into an Artificial Thymic Organoid (ATO) culture or a Feeder-Free culture.
[0064] In the ATO culture, transduced HSCs were mixed with MS5-DLL4 feeder cells to form ATOs and cultured over 8 weeks following a previously established protocol (Li et al., 2021b; Montel-Hagen et al., 2019). In the Feeder-Free culture, transduced HSCs were cultured using a StemSpan T Cell Generation Kit (StemCell Technologies) over 5 weeks following the manufacturer's instructions (Y. R. Li et al., 2022). The resulting HSC-iNKT cells isolated from ATOs or Feeder-Free culture were expanded with GC-loaded PBMCs (GC-PBMCs). To prepare GC-PBMCs, 1-1010.sup.7 PBMCs were incubated in 5 ml Cl 0 medium containing 5 g/ml GC for 1 hour, followed by irradiation at 6,000 rads. HSC-iNKT cells were mixed with irradiated GC-PBMCs at ratio 1:1, followed by culturing for 2 weeks in C10 medium supplemented with human IL-7 (10 ng/ml) and IL-15 (10 ng/ml); cell cultures were split, and fresh media/cytokines were added if needed. The resulting HSC-iNKT cell products were then collected and cryopreserved for future use.
Generation of PBMC-Derived Conventional T (PBMC-Tcon) and iNKT (PBMC-iNKT) Cells
[0065] Healthy donor PBMCs were obtained from the UCLA/CFAR Virology Core Laboratory and were used to generate the PBMC-Tc and PBMC-iNKT cells.
[0066] To generate PBMC-Tcon cells, PBMCs were stimulated with CD3/CD28 T-activator beads (ThermoFisher Scientific) and cultured in C10 medium supplemented with human IL-2 (20 ng/mL) for 2-3 weeks, following the manufacturer's instructions.
[0067] To generate PBMC-iNKT cells, PBMCs were enrich for iNKT cells using anti-iNKT microbeads (Miltenyi Biotech) and MACS-sorting, followed by stimulation with donor-matched irradiated GC-PBMCs at the ratio of 1:1 and cultured in C10 medium supplemented with human recombinnat IL-7 (10 ng/ml) and IL-15 (10 ng/ml) for 2-3 weeks. If necessary, the resulting PBMC-iNKT cells could be further purified using Fluorescence-Activated Cell Sorting (FACS) via human iNKT TCR antibody (Clone 6B11; BD Biosciences) staining.
HSC-iNKT Cell Phenotype and Functional Study
[0068] HSC-iNKT cells were analyzed in comparison with PBMC-Tcon and PBMC-iNKT cells. Phenotype of these cells was studied using flow cytometry by analyzing cell surface markers including co-receptors (i.e., CD4 and CD8), NK cell receptors (i.e., CD161, NKG2D, and DNAM-1), and memory T cell markers (i.e., CD45RO). The capacity of these cells to produce cytokines (i.e., IFN-, TNF-, IL-2, and IL-4) and cytotoxic molecules (i.e., Perform and Granzyme B) were studied using flow cytometry via intracellular staining.
Ganciclovir (GCV) In Vitro and In Vivo Killing Assay
[0069] For GCV in vitro killing assays, HSC-iNKT cells were cultured in C10 medium in the presence of titrated amount of GCV (0-50 M) for 4 days; live HSC-iNKT cells were then counted using a hematocytometer (VWR) via Trypan Blue staining (Fisher Scientific).
[0070] GCV in vivo killing assay was performed using an NSG xenograft mouse model. NSG mice received i.v. injection of 110.sup.7 HSC-iNKT cells on day 0, followed by i.p. injection of GCV for 5 consecutive days (50 mg/kg per injection per day). On day 5, mice were terminated. Multiple tissues (i.e., blood, spleen, liver, and lung) were collected and processed for flow cytometry analysis to detect tissue-infiltrating HSC-iNKT cells (identified as iNKT TCR.sup.+CD45.sup.+), following established protocols (Li et al., 2021b; Y. R. Li et al., 2022; Zhu et al., 2019).
In Vitro Tumor Cell Killing Assay
[0071] Tumor cells (110.sup.4 cells per well) were co-cultured with HSC-iNKT cells (at ratios indicated in figure legends) in Coming 96-well clear bottom black plates for 24 hours, in C10 medium. At the end of culture, live tumor cells were quantified by adding D-luciferin (150 g/ml; Caliper Life Science) to cell cultures and reading out luciferase activities using an Infinite M1000 microplate reader (Tecan).
[0072] In some experiments, 10 g/ml of LEAF purified anti-human NKG2D (Clone 1D11, Biolegend), anti-human DNAM-1 antibody (Clone 11A8, Biolegend), or LEAF purified mouse 1gG2bk isotype control antibody (Clone MG2B-57, Biolegend) was added to co-cultures, to study NK activating receptor-mediated tumor cell killing mechanism.
In Vitro Mixed Lymphocyte Reaction (MLR) Assay: Studying .sup.3rdHSC-iNKT Cell Inhibition of Allogeneic T Cell Response
[0073] PBMCs of multiple healthy donors were irradiated at 2,500 rads and used as stimulators, and non-irradiated allogeneic PBMCs were used as responders. In order to separate the different donor PBMCs when performing flow cytometry, HLA-A2.sup.+ responders and HLA-A2.sup. stimulators were used in this study. Irradiated stimulators (2.510.sup.5 cells/well) and responders (110.sup.4 cells/well) were co-cultured with or without the addition of .sup.3rdHSC-iNKT cells (110.sup.4 cells/well) in 96-well round bottom plates in C10 medium for up to 4 days. For detection of composition and phenotype using flow cytometry, cells were collected on day 1. For IFN- production using ELISA, cell culture supernatants were collected on day 4. To study CD1d-dependent killing mechanism of .sup.3rdHSC-iNKT cells, 10 g/ml of LEAF purified anti-human CD1d (Clone 51.1, Biolegend) or LEAF purified mouse lgG2bk isotype control antibody (Clone MG2B-57, Biolegend) was added to co-cultures.
Bioluminescence Live Animal Imaging (BLI)
[0074] BLI was performed using a Spectral Advanced Molecular Imaging (AMI) HTX imaging system (Spectral instrument Imaging). Live animal imaging was acquired 5 minutes after intraperitoneal (i.p.) injection of D-Luciferin (1 mg per mouse). Imaging results were analyzed using an AURA imaging software (Spectral Instrument Imaging).
Human PBMC Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Amelioration of GvHD
[0075] NSG mice were pre-conditioned with 100 rads of total body irradiation (day 1), followed by intravenous injection of 210.sup.7 healthy donor PBMCs with or without the addition of 210.sup.7 3rdHSC-iNKT cells. Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund. Various mouse tissues (i.e., blood, spleen, liver, lung, bone marrow, skin, and salivary ligand) were harvested and processed for either flow cytometry or histologic analysis.
Human PBMC Xenograft NSG Mouse Model: Studying CD14 Myeloid Cell Modulation of GvHD
[0076] NSG mice were pre-conditioned with 100 rads of total body irradiation (day 1), followed by intravenous injection of 210.sup.7 healthy donor PBMCs or 910.sup.6 CD14-depleted donor PBMCs. The amount of PBMCs given was normalized to contain the same number of T cells. Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment).
Human CD14-Depleted PBMC Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Amelioration of GvHD
[0077] NSG mice were pre-conditioned with 100 rads of total body irradiation (day 1), followed by intravenous injection of 910.sup.6 CD14-depleted donor PBMCs with or without the addition of 210.sup.7 3rdHSC-iNKT cells. Mice were weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
Raji-FG Human B Cell Lymphoma Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Retention of GvL Effect
[0078] NSG mice were pre-conditioned with 100 rads of total body irradiation (day 1), followed by subcutaneous inoculation with 110.sup.5 Raji-FG cells (day 0). On day 3, the tumor-bearing experimental mice received intravenous (i.v.) injection of 210.sup.7 healthy donor PBMCs with or without the addition of 210.sup.7 3rdHSC-iNKT cells. Tumor load were monitored over time using BLI. Mice were also weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
HL60-FG Human Acute Myeloid Leukemia Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Retention of GvL Effect
[0079] NSG mice were pre-conditioned with 175 rads of total body irradiation (day 1), followed by intravenous inoculation with 210.sup.5 HL60-FG (day 0). On day 3, the tumor-bearing experimental mice received intravenous (i.v.) injection of 210.sup.7 healthy donor PBMCs with or without the addition of 210.sup.7 3rdHSC-iNKT cells. Tumor load were monitored over time using BLI. Mice were also weighed daily, bled weekly, and scored 0-2 per clinical sign of GvHD (i.e., body weight, activity, posture, skin thickening, diarrhea, and dishevelment). Mice were terminated and analyzed when moribund.
Histological Analysis
[0080] Tissues (i.e., liver, lung, salivary glands, and skin) were collected from the experimental mice, fixed in 10% Neutral Buffered Formalin for up to 36 hours, then embedded in paraffin for sectioning (5 m thickness). Tissue sections were prepared and stained with Hematoxylin and Eosin (H&E) or anti-CD3 by the UCLA Translational Pathology Core Laboratory, following the Core's standard protocols. The H&E-stained sections were imaged on a Zeiss Observer II upright microscope. All images were captured at either 100 or 200 and processed using Zen Blue software. GvHD pathological score was calculated as follows: skin: epidermal changes (0-3), dermal changes (0-3), adipose changes (0-3); salivary: infiltration (0-4), follicular destruction (0-4); liver: duct infiltration (0-3), number of ducts involved (0-3), liver cell apoptosis (0-3); lung: infiltrates (0-3); pneumonitis (0-3), overall appearance (0-3). For CD3 surface area measurements, the anti-CD3-stained sections were scanned in their entirety using Hamamatsu Nanozoomer 2.0 HT. The % CD3.sup.+ area was determined by CD3.sup.+ area divided by total tissue area, using an Image-Pro Premier software.
Statistical Analysis
[0081] GraphPad Prism 6 (Graphpad Software) was used for statistical data analysis. Student's two-tailed t test was used for pairwise comparisons. Ordinary 1-way ANOVA followed by Tukey's multiple comparisons test was used for multiple comparisons. Log rank (Mantel-Cox) test adjusted for multiple comparisons was used for Meier survival curves analysis. Data are presented as the meanSEM, unless otherwise indicated. In all figures and figure legends, N represents the number of samples or animals utilized in the indicated experiments. A P value of less than 0.05 was considered significant. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
Discussion
[0082] iNKT cells are uniquely positioned at the crossroads of innate and adaptive immunity and have potent immunoregulatory functions in a variety of diseases (Brennan et al., 2013; Van Kaer et al., 2011). Research into harnessing iNKT cells to combat GvHD began decades ago (Lan et al., 2001), but clinical application of iNKT cells has been hindered by their scarcity in peripheral blood (Krijgsman et al., 2018). We have recently developed an ex vivo HSC-iNKT culture method that can robustly generate large quantities of pure, clonal human iNKT cells (
[0083] GvHD prophylaxis is centered around calcineurin inhibitor (CNT)-based therapy and investigations into new methods, including those depleting T cells, modulating T cell co-stimulatory pathways (e.g., checkpoints), enhancing regulatory T cells, targeting T cell trafficking, and altering cytokine pathways (Gooptu and Antin, 2021). Despite prophylactic interventions, acute GvHD is a common complication of allo-HSCT, occurring in 30-50% of patients, 14-36% of whom develop severe acute GvHD, and is a major cause of morbidity and mortality (Malard et al., 2020). The current first-line treatment for acute GvHD is systemic steroid therapy, but almost half of patients will become refractory to treatment and there is no accepted standard-of-care treatment for steroid refractory-acute GvHD (Malard et al., 2020). The dismal survival rate and poor quality of life in these patients highlight the urgent need for novel therapeutic and prophylactic agents against acute GvHD.
[0084] The driver of clinical acute GvHD is donor alloreactive T cells (Ball and Egeler, 2008). Following lymphodepletion and HSCT, host and donor antigen-presenting cells respond to host tissue damage and lead to the activation of donor T cells (Ramachandran et al., 2019). Although culpable for GvHD, HSCT-derived T cells are essential for antitumor effects, as their depletion from HSCT grafts precipitates increased relapse rates (Horowitz et al., 1990). To study the anti-GvHD potential of .sup.3rdHSC-iNKT cells, we adopted an xeno-GvHD NSG mouse model, in which human PBMCs are intravenously infused and subsequent donor T cell activation results in GvHD (
[0085] Although mechanisms are currently under investigation, our ex vivo culture of iNKT TCR transduced HSCs produces nearly all CD4.sup. HSC-iNKT cells (
[0086] iNKT cells can also play a direct role in tumor killing. Through CD1d dependent and independent means, iNKT cells have been shown to lysis a variety of tumor cells (King et al., 2018; Li et al., 2021b; Zhu et al., 2019). Furthermore, in hematological and solid tumor models, adoptive transfer of iNKT cells reduces tumor burden and enhances overall survival (Fujii et al., 2013). Our previous studies have demonstrated the antitumor functions of HSC-iNKT cells in vivo when targeting CD1d positive and negative cancer cells (Li et al., 2021b; Zhou et al., 2021). Importantly, HSC-iNKT cells do not recognize mismatched MHCs and thus pose no risk of inducing GvHD; furthermore, due to their intrinsic low expression of HLA-I and II molecules, these cells are resistant to allorejection (Li et al., 2021b; Y. R. Li et al., 2022). These features of HSC-iNKT cells make them suitable for allogeneic cell therapy.
[0087] Allo-HSCT is an established, effective treatment for hematological malignancies, but GvHD is common and debilitating adverse event for many allo-HSCT recipients. We propose to develop the off-the-shelf HSC-iNKT cell therapy to ameliorate GvHD while preserving GvL in the treatment of blood cancers. The reported ex vivo HSC-iNKT cell culture is robust and of high yield and purity, with the potential of being scaled for further translation and clinical development. From one cord blood donor, over 10,000 doses of third-party HSC-iNKT cells can be manufactured and cryopreserved for ready distribution to allo-HSCT patients; MHC matching is not needed. This study highlights the potential of .sup.3rdHSC-iNKT cells to address a critical unmet medical need and warrants further investigations of this promising off-the-shelf cell product.
Limitations of the Study
[0088] Predominant mouse models studying GvHD typically employ transplantation of T cell-depleted bone marrow and donor-derived T cells into lethally irradiated recipients; these are paramount to advance the forefront of knowledge regarding the incidence of GvHD within allo-HSCT therapeutics (Schroeder and DiPersio, 2011). In this study, healthy donor T cells were used to generate a PBMC-xenograft NSG mouse model, producing a construct where T cell-mediated GvHD could be studied and manipulated in vivo. However, limitations to this model preclude its ability to fully reflect GvHD pathology in allo-HSCT. Such complexity arises from factors such as the restricted availability of human embryonic tissue for transplant, the need for sublethal total body irradiation, demand for a high quantity of human PBMCs, and instability in the onset window of GvHD (Huang et al., 2018). Additionally, murine immunoreaction after engraftment of human immune cells is highly distinct compared to that in humans in regard to both biological phenotype and genetics. Therefore, developing a model that more accurately mimics human GvHD pathology, while reducing variance from these limitations, is necessary to understand patient reactivity to allo-HSCT therapies.
Star Methods
[0089] Detailed methods are provided in the online version of this paper and include the following: [0090] KEY RESOURCES TABLE [0091] RESOURCE AVAILABILITY [0092] Lead Contact [0093] Materials Availability [0094] Data and Code Availability [0095] EXPERIMENTAL MODEL AND SUBJECT DETAILS [0096] Mice [0097] Cell Lines and Viral Vectors [0098] Human Periphery Blood Mononuclear Cells (PBMCs) [0099] Media and Reagents [0100] METHOD DETAILS [0101] Antibodies and Flow Cytometry [0102] Enzyme-Linked Immunosorbent Cytokine Assays (ELISAs) [0103] In Vitro Generation of HSC-Engineered iNKT (HSC-iNKT) Cells [0104] Generation of PBMC-Derived Conventional T (PBMC-Tcon) and iNKT (PBMC-iNKT) Cells [0105] HSC-iNKT Cell Phenotype and Functional Study [0106] Ganciclovir (GCV)In Vitro and In Vivo Killing Assay [0107] In Vitro Tumor Cell Killing Assay [0108] In Vitro Mixed Lymphocyte Reaction (MLR) Assay: Studying .sup.3rdHSC-iNKT Cell Inhibition of Allogeneic T cell Response [0109] Bioluminescence Live Animal Imaging (BLI) [0110] Human PBMC Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Amelioration of GvHD [0111] Human PBMC Xenograft NSG Mouse Model: Studying CD14.sup.+ Myeloid Cell Modulation of GvHD [0112] Human CD14-Depleted PBMC Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Amelioration of GvHD [0113] Raji-FG Human B Cell Lymphoma Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Retention of GvL Effect [0114] HL60-FG Human Acute Myeloid Leukemia Xenograft NSG Mouse Model: Studying .sup.3rdHSC-iNKT Cell Retention of GvL Effect [0115] Histological Analysis [0116] Statistical Analysis
Star*Methods
TABLE-US-00001 KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human IFN- (ELISA, capture) BD Biosciences CAT#551221, RRID: AB_394099 Anti-human IFN- (ELISA, detection) BD Biosciences CAT#554550, RRID: AB_395472 Anti-human CD34 (Clone 581) BD Biosciences CAT#555822, RRID: AB_396151 Anti-human TCR V24-J18 (Clone 6B11) BD Biosciences CAT#552825, RRID: AB_394478 Anti-human CD45 (Clone H130) Biolegend CAT#304026, RFID: AB_893337 Anti-human TCR (Clone 126) Biolegend CAT#306716, RRID: AB_1953257 Anti-human CD4 (Clone OKT4) Biolegend CAT#317414, RRID: AB_571959 Anti-human CD8 (Clone SK1) Biolegend CAT#344714, RRID: AB_2044006 Anti-human CD45RO (Clone UCHL1) Biolegend CAT#304216, RRID: AB_493659 Anti-human CD161 (Clone HP-3G10) Biolegend CAT#339928, RRID: AB_2563967 Anti-human CD14 (Clone HCD14) Biolegend CAT#325608, RRID: AB_830681 Anti-human CD19 (Clone SJ25C1) Biolegend CAT#363005, RRID: AB_2564127 Anti-human CD11b (Clone ICRF44) Biolegend CAT#301330, RRID: AB_2561703 Anti-human CD1d (Clone 51.1) Biolegend CAT#350308, RRID: AB_10642829 Anti-human NKG2D (Clone 1D11) Biolegend CAT#320812, RRID: AB_2234394 Anti-human DNAM-1 (Clone 11A8) Biolegend CAT#338312, RRID: AB_2561952 Anti-human HLA-A2 (Clone BB7.2) Biolegend CAT#343308, RRID: AB_2561567 Anti-human IFN- (Clone B27) Biolegend CAT#506518, RRID: AB_2123321 Anti-human Granzyme B (Clone QA16A02) Biolegend CAT#372204, RRID: AB_2687028 Anti-human Perforin (Clone dG9) Biolegend CAT#308126, RRID: AB_2572049 Anti-human TNF (Clone Mab11) Biolegend CAT#502912, RRID: AB_315264 Anti-human IL-2 (Clone MQ1-17H12) Biolegend CAT#500341, RRID: AB_2562854 Anti-human IL-4 (Clone MP4-25D2) Biolegend CAT#500824, RRID: AB_2126746 Anti-human GM-CSF (Clone BVD2-21C11) Biolegend CAT#502313, RRID: AB_2561838 LEAF purified anti-human NKG2D antibody Biolegend CAT#320810, (Clone 1D11) RRID: AB_2133276 LEAF purified anti-human DNAM-1 antibody BD Biosciences CAT#559786, (Clone DX11) RRID: AB_397327 Mouse IgG1, isotype control antibody Biolegend CAT#400124, (Clone MOPC-21) RRID: AB_2890215 LEAF purified anti-human CD1d antibody Biolegend CAT#350304, (Clone 51.1) RRID: AB_10641291 LEAF purified Mouse IgG2b, k isotype ctrl Biolegend CAT#401201, (Clone MG2b-57) RRID: AB_2744505 Human Fc Receptor Blocking Solution Biolegend CAT#422302, (TrueStain FcX) RRID: AB_2818986 Mouse Fc Block (anti-mouse CD16/32) BD Biosciences CAT#553142, RRID: AB_394657 Bacterial and virus strains Lenti/iNKT-sr39TK This paper N/A Lenti/FG This paper N/A Biological samples Human peripheral blood mononuclear cells UCLA N/A (PBMCs) Cord Blood Cryo CD34 HemaCare CAT#CB34C-3 Chemicals, peptides, and recombinant proteins Streptavidin-HRP conjugate Invitrogen CAT#SA10001 IFN- (ELISA, standard) eBioscience CAT#29-8319-65 Tetramethylbenzidine (TMB) KPL CAT#5120-0053 Ganciclovir (GCV) Sigma CAT#ADV465749843 Recombinant human IL-2 Peprotech CAT#200-02 Recombinant human IL-3 Peprotech CAT#200-03 Recombinant human IL-7 Peprotech CAT#200-07 Recombinant human IL-15 Peprotech CAT#200-15 Recombinant human Flt3-Ligand Peprotech CAT#300-19 Recombinant human SCF Peprotech CAT#300-07 Recombinant human TPO Peprotech CAT#300-18 Recombinant human GM-CSF Peprotech CAT#300-03 L-ascorbic acid 2-phosphate Sigma CAT#A8960-5G B27 Supplement (50X), serum free ThermoFisher CAT#17504044 -Galactosylceramide (KRN7000) Avanti Polar SKU#867000P- Lipids 1 mg X-VIVO 15 Serum-free Hematopoietic Cell Lonza CAT#04-418Q Medium RPMI1640 cell culture medium Corning Cellgro CAT#10-040-CV DMEM cell culture medium Corning Cellgro CAT#10-013-CV Fetal Bovine Serum (FBS) Sigma CAT#F2442 MACS BSA stock solution Miltenyi CAT#130-091-376 30% BSA Gemini CAT#50-753-3079 Penicillin-Streptomycine-Glutamine (P/S/G) Gibco CAT#10378016 Penicillin:streptomycin (pen:strep) Gemini CAT#400-109 solution (P/S) Bio-products MEM non-essential amino acids (NEAA) Gibco CAT#11140050 HEPES Buffer Solution Gibco CAT#15630056 Sodium Pyruvate Gibco CAT#11360070 Beta-Mercaptoethanol Sigma SKU#M6250 Normocin InvivoGen CAT#ant-nr-2 Cell Fixation/Permeabilization Kit BD Biosciences CAT#554714 RetroNectin recombination human fibronectin Takara CAT#T100B fragment, 2.5 mg 10% neutral-buffered formalin Richard-Allan CAT#5705 Scientific D-Luciferin Caliper LIfe CAT#XR-1001 Science Isoflurane Zoetis CAT#50019100 Phosphate Buffered Saline (PBS) pH 7.4 (1X) Gibco CAT#10010-023 Formaldehyde Sigma-Aldrich CAT#F8775 Golgistop Protein Transport Inhibitor BD Biosciences CAT#554724 Phorbol-12-myristate-13-acetate (PMA) Calbiochem CAT#524400 Ionomycin, Calcium salt, Calbiochem CAT#407952 Streptomyces conglobatus Poloxamer Synperonic F108 Sigma CAT#07579-250G- F Prostaglandin E2 Cayman Chemical CAT#14-190-136 Fixable Viability Dye eFluor506 affymetrix CAT#65-0866-14 eBioscience Critical commercial assays Human CD34 MicroBeads Kit Miltenyi Biotec CAT#130-046-703 Human CD14 MicroBeads Kit Miltenyi Biotec CAT#130-050-201 Human Anti-iNKT MicroBeads Miltenyi Biotec CAT#130-094-842 Fixation/Permeabilization Solution Kit BD Sciences CAT#55474 StemSpan Lymphoid Differentiation Coating Stem Cell CAT#9925 Material (100X) Technologies StemSpan SFEM II Stem Cell CAT#9605 Technologies ImmunoCult Human CD3/CD28/CD2 T Cell Stem Cell CAT#10970 Activator Technologies Cryostor cell cryopreservation media Sigma CAT#C2874- 100 ML Experimental models: Cell lines Human Burkitts lymphoma cell line Raji ATCC CCL-86 Human acute myeloid leukemia cell line HL60 ATCC CCL-240 Human Burkitts lymphoma cell line Raji-FG This paper N/A Human acute myeloid leukemia cell This paper N/A line HL60-FG Experimental models: Organisms/strains NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) The Jackson Stock #: 005557 Laboratory Recombinant DNA Vector: parental lentivector pMNDW Giannoni et N/A al., 2013 and Lan et al., 2006 Software and algorithms FlowJo Software FlowJo https://www.flowjo.com/solutions/flowjo/downloads Living Imaging 2.50 software Xenogen/PerkinElmer http://www.perkinelmer.com/lab- products-and-services/resources/in- vivo-imaging-software-downloads.html AURA imaging software Spectral https://spectralinvivo.com/software/ Instruments Imaging I-control 1.7 Microplate Reader Software Tecan https://www.selectscience.net/tecan/i- control-microplate-reader-software/81307 ImageJ ImageJ https://imagej.net/Downloads Prism 6 Graphpad https://www.graphpad.com/scientific- software/prism/
Keywords
[0117] Invariant natural killer T (iNKT) cells; Hematopoietic stem cell (HSC) engineering; Ex vivo T cell differentiation; Graft versus host disease (GvHD); Graft versus leukemia/lymphoma (GvL) effect
Supplemental Information
[0118] Supplemental Information discussed herein (e.g.,
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[0191] All publications mentioned herein (e.g. the references numerically listed above and U.S. Patent Applications having serial numbers U.S. Pat. No. 10,927,160, U.S. Ser. No. 17/251,118 and U.S. Ser. No. 17/618,240) are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications. Certain aspects and embodiments of the invention are found in the disclosure of the Appendix submitted herewith, which is herein incorporated by reference.