Patent Application
20240287194
Provided herein are compositions and methods for of treating a subject who has a solid tumor or who has a viral infection, or promoting cytotoxicity of natural killer (NK) cells in a subject, using (i) blocking antibodies to ECM components and/or (ii) inhibitors of collagen deposition or production.
It has long been appreciated that downregulation of Major Histocompatibility Complex Class I (MHC-I) is a primary mechanism for immune evasion utilized by cancer cells (1). In the era of cancer immunotherapeutics, MHC-I downregulation has emerged as a major resistance mechanism against immunotherapy as malignant cells escape from CD8+ T cell detection and elimination (1). However, the pervasive selection of this immune evasion strategy by cancer cells is perplexing in that MHC-I downregulation is expected to render cells vulnerable to Natural killer (NK) cell killing (2-4). The dilemma of NK cells' role in controlling cells that fail to express self MHC-I in vivo was originally encountered in the transplantation field. In the context of hybrid resistance, parental skin grafts are accepted, but bone marrow (BM) cells are rejected by F1 hybrids in an NK cell-dependent manner (5, 6). Subsequent studies have shown that NK cells in wild-type (WT) syngeneic recipients reject donor MHC-I-deficient BM cells, while MHC-I-deficient solid organ transplants are not rejected (7, 8). Elucidating the mechanism that underlies tissue-specific NK cell responses will have major implications in cancer immunology, transplant biology and virology.
NK cells belong to the innate lymphoid cell (ILC) family that reside in most tissues and form a swift acting innate barrier against viral infections and cells undergoing malignant transformation (9). Conventional NK (cNK) cells, defined by expression of NK1.1, NKp46 and CD49b in mice, are found in circulation, secondary lymphoid organs and most other tissues (9). Another member of the ILC family that is closely related in phenotype and function is the tissue-resident NK (trNK) or ILC1 cell that lacks CD49b but expresses CD49a (9). These cells are a non-migrating, NK cell-like population (10) that are thought to develop from two distinct pathways: 1) differentiation from the innate lymphoid cell progenitor (ILCP) (11) or 2) differentiation from cNK cells in the presence of TGFβ (12, 13) in a non-mutually exclusive manner.
Provided herein are methods for treating a subject who has a solid tumor and/or a viral infection. The methods comprise administering to the subject a therapeutically effective amount of (i) blocking antibodies to ECM components and/or (ii) inhibitors of collagen deposition or production.
Also provided are methods for promoting cytotoxicity of natural killer (NK) cells in a subject. The methods comprise administering to the subject a therapeutically effective amount of (i) blocking antibodies to ECM components and/or (ii) inhibitors of collagen deposition or production.
Also provided are compositions comprising (i) blocking antibodies to ECM components and/or (ii) inhibitors of collagen deposition or production, for use in a method of treating a subject who has a solid tumor, a viral infection, and/or for promoting cytotoxicity of natural killer (NK) cells in a subject.
In some embodiments, the blocking antibodies to ECM components bind to LAIR1/2; Cd29 (integrin R); CD49A; GPR561; integrin αV or integrin β3.
In some embodiments, the inhibitor of collagen deposition or production is losartan or 3,4-dihydroxybenzoic acid (DHB).
In some embodiments, the subject has breast, prostate, pancreatic, brain, hepatic, lung, kidney, skin, or colon cancer. In some embodiments, the skin cancer is melanoma.
In some embodiments, the subject has a viral infection selected from a respiratory virus (optionally influenza viruses (A and B), H5N1 and H7N9 avian influenza A viruses, parainfluenza viruses 1 through 4, adenoviruses, respiratory syncytial virus A and B and human metapneumovirus, rhinoviruses, or coronaviruses); a gastrointestinal virus (optionally rotavirus, norovirus, astrovirus, adenovirus 40 and 41, or coronavirus-like agents, or enteroviruses, optionally coxsackieviruses and echoviruses); hepatitis A, B, C, D, and E viruses; arboviruses, arenaviruses, and filoviruses; and viruses that infect the skin or mucosal membranes (optionally herpes simplex viruses (HSV), papillomavirus, polyomavirus, and poxviruses).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
B2M gene alteration frequency in various solid cancers and leukemias from the TCGA pan-cancer dataset and other public cancer databases from eBioPortal.
Full NK cell activation against a target cell is accomplished by the integration of multiple inhibitory and activation signals, a process that limits indiscriminate killing of healthy host cells. Self MHC-I molecules function as the dominant inhibitory signal by binding NK cell-expressed Ly49 receptors in mice, or killer inhibitory receptors (KIR) in humans, to prevent degranulation and cytokine release. Multiple activation signals, together with downregulation or loss of inhibitory signals, are required to enable complete NK cell cytotoxicity (3, 14, 15). Following their licensing to activate their cytotoxic program (16-18), NK cells destroy the target cells by production and release of granzymes and perforin in the immunological synapse (19). Fas-FasL and TRAIL are also used by NK cells for target cell elimination (20).
Although in vitro models of NK cell activation have provided insights into signaling requirements that initiate NK cell killing of target cells, the conditions in vivo are much more complex in that non-MHC ligands and potentially unknown inhibitory and activation pathways of varying strengths, acting together with cytokines, set the NK cell activation threshold (21). Most in vivo NK cell activation models have focused on NKG2D and other activating ligands and cytokines that also activate CD8+ T cells (22), complicating interpretation of NK versus CD8+ T cell effects. To investigate the mechanism of NK cell activation in peripheral tissues, we utilized the NK cell-specific m157-Ly49H activation axis in a skin transplantation model. m157 is murine cytomegalovirus (MCMV)-encoded glycoprotein expressed on the surface of infected cells. This MHC-I-like molecule serves as an NK cell-specific activation signal, directing the targeted killing of MCMV infected cells through recognition by the Ly49H receptor, which is only present on the surface of NK cells on the C57BL/6 (B6) background (23). As a result, B6 mice are resistant to MCMV infection, while BALB/c and other strains are susceptible (24). Thus, the m157-Ly49H axis can be used to specifically study NK cell activation and its downstream function in vivo.
Herein, we demonstrate that upon exit from the circulation into the skin graft in response to m157, NK cells are exposed to extracellular matrix (ECM) proteins, which can block NK cell cytotoxicity while promoting its helper function. NK cell activation induced by the combination of m157 and missing self MHC-I (25) resulted in skin graft rejection in an NK and T cell-dependent manner. RNA-Seq analysis revealed significant downregulation in cytotoxic mediators together with upregulation in inflammatory cytokines and chemokines, when comparing cNK cells that entered the skin with circulating cNK cells. We found that in vitro exposure to ECM components, collagens and elastin, transformed the function and signaling within cNK cells. Furthermore, control of circulating melanoma cells occurred in an NK cell-dependent manner, yet the growth of these same melanoma cells subcutaneously was not impacted by NK cells embedded in ECM. Importantly, blocking collagen deposition in subcutaneous melanomas led to an NK cell-mediated tumor suppression. Human solid cancers, but not leukemias, could afford to downregulate MHC-I to escape CD8+ T cell-mediated elimination, which suggested that NK cells within peripheral tissues, unlike those in circulation, lacked a direct cytotoxic function. This study reveals a fundamental aspect of NK cell biology, which governs the interplay of NK cells with cancer, organ transplantation and viral infection.
NK cell killing of virally infected or malignant cells is a vital aspect of innate immunity. Yet, as noted above the mechanisms behind the inability of NK cells to directly eliminate NK cell-sensitive targets in peripheral tissues have remained undetermined. We show here, using skin transplantation and cancer models, that once NK cells exit the circulation and enter the target tissue, they experience a profound change in their function (
Large numbers of NK cells are recruited into m157-expressing donor skin; however, NK cells are unable to directly reject the skin grafts even with the loss of MHC-I and the addition of NK cell-stimulating poly(I:C) and cytokines in a syngeneic model. This demonstrates that the MHC-I-independent inhibition of NK cell's cytotoxicity dominated over multiple strong activation signals tested. Skin graft rejection is accomplished using F1 recipients, which provides missing-self for NK cell activation while simultaneously preserving MHC-I expression on the donor cells. In this system, NK cells licensed by BALB/c MHC-I are fully activated against B6 MHC-I-expressing m157+ donor skin cells (i.e., missing-self) (25) while CD8+ T cells are able to contribute to the rejection as the MHC-I signaling axis remains operational. Graft rejection begins after day 12 post-transplant, which is approximately 7 days later than the initial accumulation of NK cells in the graft. The majority of rejection is completed by day 20 post-transplant, which is in agreement with the timing of an adaptive immune response. The timing of NK and T cell infiltration into the skin graft, the kinetics of graft rejection and the requirement for both NK and T cells in this process suggest that NK cells activate the adaptive immune response and T cells function as effectors to reject m157tg donor skin grafts in F1 recipients.
NK cell activation has been associated with the production of high levels of IFNγ and TNFα in addition to CCL4, CCL5, XCL1, and GM-CSF (44-46). These cytokines and chemokines promote macrophage activation together with recruitment and activation of DCs and T cells at sites of inflammation (47-51). Interestingly, activated cNK cells entering the skin from circulation immediately upregulated the CXCR3 ligands, CXCL9 and CXCL10, in addition to CCL2 and CXCL5, chemokines that profoundly induce T cell recruitment. In contrast, CCL5 was downregulated and CCL4, XCL1, and GM-CSF expression were not altered in cNK cells as they exit the circulation. A transient increase in CCR7 is also observed, which may drive newly recruited cNK cells deeper into the skin, and subsequently the draining lymph nodes, where CCL21+ lymphatic endothelial cells reside. A concomitant reduction in the expression of T-bet and EOMES and an increase in ICOS may drive a helper cNK cell functional program. Furthermore, the cytotoxic effectors Gzma, Gzmb and Prf1 were either reduced or not changed in activated cNK cells as they exit the circulation, supporting observations of limited NK cell direct cytotoxicity in the skin. Without wishing to be bound by theory, although NK cells may directly activate CD8+ T cells in the skin graft, an indirect path through dendritic cells and/or CD4+ T cell activation likely plays an important role in the observed NK cell helper function, which will be further investigated in future studies (51-53). While direct killing of some target cells by NK cells may occur, the rejection of m157tg skin graft is driven by CD8+ and CD4+ cells, which likely recognize m157 as foreign antigen expressed by the donor skin cells (54-56).
Previous studies have shown a role for TGFβ in impairing NK cell function in cancer models by promoting the conversion of cNK cells to trNK cells (12, 13, 37). As shown herein, the impairment in NK cell cytotoxicity is an immediate consequence of cNK cell exit from the circulation into a peripheral tissue microenvironment. In response to skin transplantation, CD49a CD49b trNK cells develop from cNK cells that have exited the circulation and populated the skin graft over time. Interestingly, cNK cells upregulated TGFβ receptor as soon as they exited circulation, which may promote their conversion into trNK cells in the TGFβ-rich environment of the skin graft. Although they may provide cytokine-mediated help to T cells later in the response, trNK cells are not seen in significant numbers until after day 14 post-transplant. In addition, blocking trNK cell development by deleting Tgfbr2 (12, 13) or Hobit transcription factor (38) does not result in NK cell cytotoxicity or graft rejection. These findings demonstrate that the switch in NK cell function from killer to helper is an immediate consequence of its exit from circulation and entry into the peripheral tissue microenvironment.
Cancers in peripheral tissues that are expected to be strong targets for NK cells have often failed to show objective responses (57-59). These results suggest that NK cells experience inhibition of their cytotoxicity likely mediated by collagens and elastin once they exit the circulation and enter the stroma. This may explain the distinct selection for loss of MHC-I by solid cancers but not leukemias in humans.
Described herein are methods for promoting cytotoxicity of NK cells in peripheral tissues by inhibiting the interaction of NK cells with components of the ECM. These methods are useful, e.g., in treating solid tumors and viral infections.
The present methods include administration of therapeutically effective amounts of blocking antibodies to ECM components and/or inhibitors of collagen deposition. In some embodiments, the administration is local administration, e.g., by injection or infusion into or near a tumor, or systemic administration, e.g., by intravenous injection or infusion.
The methods described herein include methods for the treatment of cancer. In some embodiments, the cancer is a solid tumor, e.g., breast, prostate, pancreatic, brain, hepatic, lung, kidney, skin, or colon cancer. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
As used in this context, to “treat” means to ameliorate at least one symptom of the disorder. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with abnormal apoptotic or differentiative processes will result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and/or decreased morbidity and mortality, inter alia.
Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
In some embodiments, the cancer is not a hematopoietic neoplastic disorder. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
Methods for identifying and diagnosing subjects with cancer, e.g., with solid tumors, are known in the art.
The methods described herein include methods for the treatment of viral infections, to boost NK cytotoxicity in virally infected subjects. In some embodiments, the viral infection is an infection with a respiratory virus, e.g., influenza viruses (A and B), H5N1 and H7N9 avian influenza A viruses, parainfluenza viruses 1 through 4, adenoviruses, respiratory syncytial virus A and B and human metapneumovirus, rhinoviruses, or coronaviruses; a gastrointestinal virus, e.g., rotavirus, norovirus, astrovirus, adenovirus 40 and 41, or coronavirus-like agents, as well as enteroviruses, which include coxsackieviruses and echoviruses; hepatitis A, B, C, D, and E viruses; arboviruses, arenaviruses, and filoviruses; viruses that infect the skin or mucosal membranes like herpes simplex viruses (HSV), papillomavirus, polyomavirus, and poxviruses. Topical/local or systemic administration can be used as appropriate. For example, to treat infections in the lungs, administration by inhalation or insufflation, e.g., of an aerosol or fine powder, can be used; for infections of skin or mucosal membranes, topical delivery method can apply. Systemic administration can also be used.
Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment, e.g., who has been identified as having a viral infection as described herein. Methods for identifying and diagnosing subjects with viral infections are known in the art. In some embodiments, the treatment is administered systemically, e.g., parenterally or orally; in some embodiments, e.g., where a specific organ or tissue is infected, the treatment is administered more locally or directly to the affected tissues, e.g., topically, vaginally, rectally, orally, ocularly, or by inhalation.
The present methods include the use of blocking antibodies to ECM components, including antibodies to LAIR 1/2; CD29 (integrin R); CD49A; GPR561 and integrin αV or β3. The methods include the administration of these antibodies (or combinations thereof, e.g., combination of lair1 plus CD29 (CD49b and CD49a) antibodies), or fusion proteins comprising the antibodies (or combinations thereof), that block ECM protein/receptor interaction. For example, binding and functional assays of NK cell activation and target killing in the presence of ECM proteins +/−antibody can be used to determine the efficacy of antibody blockade.
The term “antibody” as used herein refers to an immunoglobulin molecule or an antigen-binding portion thereof. Examples of antigen-binding portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments, which retain the ability to bind antigen. The antibody can be polyclonal, monoclonal, recombinant, chimeric, de-immunized or humanized, fully human, non-human, (e.g., murine), or single chain antibody. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. Methods for making antibodies and fragments thereof are known in the art, see, e.g., Harlow et. al., editors, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice, (N.Y. Academic Press 1983); Howard and Kaser, Making and Using Antibodies: A Practical Handbook (CRC Press; 1st edition, Dec. 13, 2006); Kontermann and Dübel, Antibody Engineering Volume 1 (Springer Protocols) (Springer; 2nd ed., May 21, 2010); Lo, Antibody Engineering: Methods and Protocols (Methods in Molecular Biology) (Humana Press; Nov. 10, 2010); and Dübel, Handbook of Therapeutic Antibodies: Technologies, Emerging Developments and Approved Therapeutics, (Wiley-VCH; 1 edition Sep. 7, 2010).
Exemplary blocking antibodies are shown in the following table A.
Other antibodies that block ECM components can also be identified, e.g., using binding assays and functional assay of NK cell activation and target killing in the presence of ECM proteins +/−antibody to determine the efficacy of antibody blockade.
The methods described herein can also include administering an inhibitor of collagen deposition or production, including losartan (e.g., losartan potassium) or 3,4-dihydroxybenzoic acid (DHB), LOX inhibitors (e.g., 2-aminopropionitrile), LOXL2 inhibitors (e.g., ellagic acid, PXS-5153A), antifibrotic drugs (e.g., Pirfenidone or Nintedanib, or Ormeloxifene); or TGFb inhibitors (e.g., SB431542).
An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect, e.g., boosting NK cell cytotoxicity to increase anti-tumor and anti-viral immunity in a subject. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments.
Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The methods described herein include the use of pharmaceutical compositions comprising or consisting of (1) blocking antibodies to ECM components and/or (2) inhibitors of collagen deposition or production, as active ingredients. In some embodiments, the compositions include a combination of lair1 plus CD29 (CD49b and CD49a) antibodies.
Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.
Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
The following materials and methods were used in the Examples set forth below.
C57BL/6 albino WT, B2m, m157tg, m157tg, B2m−/−, C57BL/6 Ncr1iCre, ROSAmT-mG, Ncr1iCre, Tgfbr2fl/fl, Act-mOVAtg, C57BL/6×BALB/c F1 Ncr1iCre, ROSAmT-mG, C57BL/6 Ncr1iCre, ROSADTR, C57BL/6×BALB/c F1 Ncr1iCre, ROSADTR and C57BL/6×BALB/c F1 CD4Cre, ROSADTR were bred in house. C57BL/6 mice (Charles River, Wilmington, MA, USA, strain code: 207) were used in the parabiosis, LEGENDplex and tumor experiments. C57BL/6-Ly5.1 mice (Charles River) were used for in vivo BM rejection experiments. C57BL/6 Hobit−/− mice and Lair1−/− mice were kind gifts from KP van Gisbergen, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, Amsterdam, The Netherlands, and Svetlana Komarova, Faculty of Dentistry, McGill University, Quebec, Canada, respectively. Mutant mice were genotyped using the primers listed in table 1. All mice were housed under specific pathogen-free conditions, given water and food ad libitum, in the animal facility at Massachusetts General Hospital in accordance with animal care regulations. All mice were closely monitored by the authors, facility technicians and an independent veterinarian when necessary. All procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Massachusetts General Hospital.
C57BL/6 albino ear skin from WT, B2m−/−, m157tg, m157tg,B2m−/− and Act-mOVAtg mice was harvested after euthanasia, cartilage was removed and skin placed in a petri dish with 1×PBS (Life Technologies, Thermo Scientific, Grand Island, NY, USA, catalog no. 14190144). Recipient mice were anesthetized for the procedure with an intraperitoneal (i.p.) injection of 4.5 μL/g body weight of 10 mg/mL ketamine (Keta Ved®, Boehringer, Ingelheim Vetmedica, Fort Dodge, IA, USA, catalog no. 045-290) combined with 0.5 mg/mL xylazine (AnaSed®, Akorn, Lake Fortes, IL, USA, catalog no. 139-236) sterile solution in addition to isofluorane (Baxter Healthcare Corporation, Deerfield, IL, USA, catalog no. 1001936040) as required. Mice were placed on a heating pad during and after the surgery. Backs of recipient mice were shaved and cleansed with 70% ethanol. A circular piece of recipient skin approximately 1.5 times the area of ear skin to be transplanted was excised from the back of recipient mice and subcutaneous fascia was removed. Donor ear skin was placed split side down and sutured using n° 6-0 silk surgical sutures (Ethicon, Puerto Rico, catalog no. K889H). Approximately one dozen equidistant sutures were made around the donor skin to attach the ear to the recipient tissue. Next, the skin transplant was coated with antibiotic ointment (Medline Industries, Northfield, IL, USA, catalog no. CUR001231) and protected with a 2 cm×2 cm patch of Vaseline gauze (Convidien, Mansfield, MA, USA, catalog no. 884413605). A bandage (Tensoplast®, BSN medical, Charlotte, NC, USA, catalog no. 02115-00) was wrapped around the torso of the mouse and sutured in place using n° 4-0 silk surgical sutures (Ethicon, Puerto Rico, catalog no. K871) on the dorsal region suturing into the back skin adjacent to the shoulders and hips. Seven days post-surgery the bandage was removed, and donor skin was monitored daily for any sign of inflammation and/or rejection. Pictures were taken every other day and ImageJ (Version 1.52a) was used to measure graft size. Recipient and donor mice were sex and aged-matched.
Mice were anesthetized using ketamine/xylazine and 1 μg of monoclonal anti-CD45-BV605 (clone 30-F11, BioLegend, San Diego, CA, catalog no. 103155) was injected by retroorbital injection to label circulating CD45+ cells. After three minutes had elapsed, peripheral blood was collected by retroorbital bleeding and, following euthanasia, liver, spleen, lymph nodes, donor and recipient skin were collected for analysis. Red blood cells in peripheral blood, spleen and liver were lysed using red blood cell lysis buffer (RBC lysis buffer 10X, Biolegend, catalog no. 420301). After washing with 1×PBS/2% FCS/5 mM EDTA, 5×106 cells were prepared for flow cytometry staining.
The back of the mouse was shaved and a large rectangle of shaved skin encompassing the donor graft and recipient skin was excised. Large regions of fat on the underside of the skin were carefully scraped away then the donor and recipient skin were separated with a razor blade. These were placed in separate dishes and chopped with scissors into ˜1 mm pieces. Each sample was then placed into a 15 mL tube with 10 mL digestion buffer (RPMI 1640 (Life Technologies, catalog no. 21870076), 200 U/mL Collagenase IV (Worthington Biochemical, Lakewood, NJ, USA)). Skin was incubated for 2 hours at 37° C. with shaking. Following incubation, digested skin was poured onto a 70 μM strainer placed in a 50 mL tube. The 15 mL tube was rinsed with ˜3 mL of 1×PBS/2% FCS/5 mM EDTA and added to the digested sample. Skin was mashed with a plunger through the 70 μM strainer which was then rinsed ˜3 times with a small volume of 1×PBS/2% FCS/5 mM EDTA. The cells were centrifuged at 300 g for 5 minutes at 4° C. and resuspended in 2.4G2 blocking solution before flow cytometry staining. NK cells were stained for activating, inhibitory receptors and CD107a surface expression for baseline evaluation. For the ex vivo NK cell stimulation assay, donor skin graft derived cells were resuspended in RPMI/10% FBS/1% penicillin and streptomycin (P/S, Thermo Fisher, Waltham, MA, catalog no. 14140122)/20 mM Glutamine and were plated on control IgG (clone MOPC-173, Biolegend, catalog no. 400203) or aNK1.1 (clone PK136, Biolegend, catalog no. 108703) pre-coated plate (Nunc™ MaxiSorp™ plate, Biolegend, catalog no. 423501) or treated with 1×PMA/Ionomycin solution (Biolegend, catalog no. 423301). The cells were incubated at 37° C./5% CO2 for total 4 hours with last 3 hours in the presence of 1 μL of anti-CD107a-BV421 (1D4B, Biolegend, catalog no. 121618), 1×Brefeldin A (Biolegend, catalog no. 420601) and 1×Monensin (Biolegend, catalog no. 420701). Following the co-culture, cells were washed with 1×PBS/2% FCS/5 mM EDTA and the pellet was resuspended in 2.4G2 blocking solution before flow cytometry staining.
The peritoneal cavity was opened to expose the liver and 5-10 mL of 1×PBS was injected into the hepatic portal vein using a 27G needle to perfuse the liver. The gall bladder was then removed and all lobes of the liver collected and mashed through a 70 μm filter sitting in a 50 mL tube using a syringe plunger. The filter was rinsed with 1×PBS/2% FCS/5 mM EDTA 3-4 times during the mashing procedure. The cell suspension was centrifuged at 300 g for 5 minutes at 4° C. then supernatant aspirated and the cell pellet washed with 10 mL of 1×PBS/2% FCS/5 mM EDTA. Following centrifugation and aspiration of supernatant the pellet was re-suspended in 25 mL of isotonic Percoll (8.44 mL Percoll [Healthcare Biosciences, Uppsala, Sweden, catalog no. 17-0891-01]/0.47 mL 20×PBS/16.09 mL 1×PBS) at room temperature (RT). Liver cell suspensions were centrifuged at 693 g for 12 minutes at RT with no brake, the leukocyte pellet at the bottom of the tube was then washed with 10 mL of 1×PBS/2% FCS/5 mM EDTA and red blood cells lysed (RBC lysis buffer 10X, BioLegend, catalog no. 420301). Following washing with 1×PBS/2% FCS/5 mM EDTA the pellet was resuspended in 2.4G2 blocking solution before flow cytometry staining.
Single-cell suspensions from all samples were prepared by straining through a 70 μm filter. 2.4G2 supernatant treated cells were stained in 1×PBS/2% FCS/5 mM EDTA with the appropriate surface antibodies (Table 2) for 30 minutes at 4° C., washed and analyzed by flow cytometry. For intracellular staining, cells were fixed and permeabilized using True-Nuclear Transcription Buffer set (Biolegend, catalog no. 424401) according to the manufacturer's protocol then incubated with appropriate antibodies (Table 2) for 60 min at RT, washed and analyzed by flow cytometry. For in vitro signaling experiments, following the specified time points, cells were fixed with an equal volume of pre-warmed 1×aldehyde based fixation buffer (Biolegend, catalog no. 420801) for 20 min at RT. Fixed cells were permeabilized by drop-wise addition of chilled methanol-based solution True-Phos Perm Buffer (Biolegend, catalog no. 425401) while mixed on a vortex. Cells were stored in-20° C. and dark for 1-3 days before washing them two times with ˜3 mL of 1×PBS/2% FCS/5 mM EDTA. Cells were subsequently stained with appropriate antibodies against surface markers and specific phosphorylated signaling proteins (Table 2) for 60 min at RT, washed and analyzed by flow cytometry. Cells were assayed on a BD LSRFortessa X-20 flow cytometer (BD Bioscience, Billerica, MA, USA) and data were analyzed using FlowJo software Version 10 (Tree Star, Ashlad, OR, USA). NK cells in WT non-reporting mice were identified as CD3− NK1.1+NKp46+.
Bone marrow cells from B6-Ly5.1 (CD45.1), B6 WT (CD45.2) and B6 B2m−/− (CD45.2) were collected from the tibia and femur by flushing bones with RPMI 1640 into a petri dish under sterile conditions. Cells were then counted and resuspended in RPMI 1640. Ly5.1:WT (control) and Ly5.1:B2m−/− (test) BM cells were mixed 1:1 and intravenously injected into sub-lethally irradiated (450 cGy) Ncr1icre, RosamT-mG recipients. Three days later, spleens of recipient mice were harvested and the ratio WT:Ly5.1 and WT:B2m−/− cells was determined by flow cytometry. % rejection was calculated as follows:
m157tg, B2m/donor skin was transplanted onto the back of C57BL/6 WT mice following the above-mentioned skin transplant procedure. On day one post-transplant, six spleens from Ncr1iCre ROSAmTmG were harvested and mashed through a 70 μm filter. Spleens were resuspended in RBC lysis for two minutes and washed with 1×PBS/2% FCS/5 mM EDTA. NKp46-GFP− ROSA cells were sorted using a Sony FX500 cell sorter (Sony Biotechnology, San Jose, CA, USA). Sorted cells were centrifuged and 8.5×105 NKp46-GFP+ cells resuspended in 200 μL of sterile RPMI 1640 were filtered through a 40 μm strainer followed by intravenous injection into recipient mice using a sterile 28G syringe (0.5 mL BD insulin syringe, Franklin Lakes, NJ, USA, catalog no. 329461). Negative controls consisted of mice injected with 200 μL RPMI 1640 alone. 20 days post-transplant, mice were euthanized and peripheral blood, spleen, liver, donor and recipient skin were collected for analysis.
Parabiont partners were co-housed in the same cage for a week before the surgery. Eight to twelve week old female C57BL/6 WT mice were surgically connected to Ncr1iCre ROSAmTmG weight and age-matched partner female mice. Surgery was performed on a heating pad and animals anesthetized using isoflurane. Longitudinal skin incisions were made on the shaved sides of each animal. Knee and elbow joints from each animal were first sutured using n° 4-0 surgical suture. Following attachment of joints, the skin of the animals was connected using n° 6-0 sutures ending with a double surgical knot. To minimize pain, 0.1 mg of Carpofren (Rimadyl, Zoetis, Brazil, catalog no. 141-199) was injected I.P. each day for two days post-surgery, in addition to close monitoring every day for signs of pain or stress. On day 21 post-parabiosis, ear skin from m157tg B2m−/− was transplanted onto the back of the C57BL/6 WT mouse of each pair. After 20 days, mice were euthanized and peripheral blood, spleen, liver, donor and recipient skin from the C57BL/6 WT parabiont were collected for analysis.
At day 20 post m157tg B2m−/− transplantation onto Ncr1iCre ROSAmTmG B6 mice and day 10 post m1574 g transplantation onto Ncr1iCre ROSAmTmG F1 mice, NK cells from blood, spleen and donor skin (cNK and trNK) and recipient skin (trNK cells) were sorted for RNA-sequencing. First, mice were anesthetized using ketamine/xylazine and 1 μg of monoclonal anti-CD45-BV605 (clone 30-F11, BioLegend) was injected via the retroorbital route three minutes before tissue harvest to label circulating CD45+ cells. Tissues were harvested as described above and, following staining, NKp46+ROSA−CD45+CD49a−CD49b+ (cNK) from peripheral blood and spleen, CD3−NKp46+ROSA−CD45−CD49a−CD49b+ (cNK), CD3− NKp46+ROSA−CD45−CD49a+CD49b+ (dpNK), CD3−NKp46+ROSA−CD45−CD49a+CD49b− (trNK) from donor skin and CD3−NKp46+ROSA−CD45−CD49a+CD49b− (trNK) cells from recipient skin were sorted using a BD FACSAria II (BD Bioscience, Billerica, MA, USA). Sorted NK cells were collected in 15 mL tubes containing 3 mL of RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS, Corning, Manassas, VA, USA) and 5% P/S.
Sorted cells were centrifuged, media aspirated and resuspended in 5 μL of TCL buffer with 1% β-mercaptoethanol (BME, Fisher Scientific, catalog no. 21-985-023) and added to a 96-well Eppendorf twin-tec barcoded plate (Eppendorf, NY, USA). Plates were stored at −80° C. until sequencing as described (60). Modified SmartSeq2 cDNA and Illumina Nextera XT library construction and sequencing were conducted at the Broad Institute of MIT and Harvard using an Illumina NextSeq 500 System (Boston, MA, USA). All samples were quasi-mapped to GRCm38 (mm10) using Salmon with the “gcBias” and “seqBias” options and collapsed down to gene-level abundance estimates using the “EnsDb.Mmusculus.v79” annotation package. All downstream differential expression analysis was carried out using DESeq2. The results table was restricted to genes with a minimum of 10 total counts across the dataset, and mitochondrial, pseudo-, and ribosomal genes were removed. Original data are available at the NCBI Gene Expression Omnibus (GEO), accession numbers: GSE148600. Gene set enrichment analysis (GSEA) (broad.mit.edu/gsea/) was used to identify significantly up- or down-regulated pathways in CD49b+ cNK cells in donor skin in comparison with CD49+ cNK cells in circulation (blood and spleen). GSEA analysis was performed using MSigDB 50 hallmark gene sets (h.all) and the curated 6226 gene sets (c2.all) and pathways containing enriched genes between 15 and 1000 genes were considered.
B16-F10 wildtype (WT) and B2m−/− cell lines were maintained in RPMI 1640 media supplemented with 10% FBS and 1% P/S and cultured at 37° C./5% CO2. WT Mouse Embryonic Fibroblasts (WT-MEF) and m157-expressing MEF (m157-MEF) were a gift from Wayne Yokoyama, Washington University, St Louis, MO, USA. MEFs were cultured in RPMI 1640 media, containing 10% FBS, 1% P/S/20 mM Glutamine (Life Technologies, catalog no 25030-081).
All tumor experiments were performed in eight to twelve-week-old C57BL/6 female WT or Ncr1iCre ROSAmT-mG mice. For the S.C. tumor model, 2.5×105 B16-F10 WT or B2m−/− cells were subcutaneously injected into the shaved flanks of mice. Tumor growth was measured every other day using a digital caliper starting from day seven. Tumor volume was calculated as follows:
To inhibit collagen deposition in and around the tumor, mice were administered with 60 mg/kg of Losartan Potassium (Fisher Scientific, catalog no. L02325G) or 40 mg/kg of 3,4-dihydroxybenzoic acid (DHB) (Sigma-Aldrich, catalog no. 37580) in 100 μL of sterile 1×PBS while the control mice were administered with the equivalent volume of sterile 1×PBS. These drugs were injected I.P. every day for the duration of the study starting the day of tumor inoculation.
For the collagenase/hyaluronidase experiment, 2.5×105 B16-F10 B2m−/− cells were resuspended in 1×collagenase and hyaluronidase mix (Stemcell, catalog no. 07912) and were subcutaneously injected into the shaved flanks of mice. To deplete collagen within the tumor, mice were administered with 1×collagenase and hyaluronidase mix (Stemcell, catalog no. 07912) in 100 μL of sterile 1×PBS while control mice were administered with the equivalent volume of sterile 1×sPBS. These enzymes were injected via intratumoral (I.T.) route starting on the day of tumor inoculation (day 0), day 1, day 2, day 3, day 4 and then every other day (day 6, 8, 10, etc.) until the end of the study. Except for day 3 tumor harvest, mice were euthanized when a tumor reached 2 cm in diameter in accordance with IACUC protocols. For early time point tumor analysis, specific Ncr1iCre, ROSAmT-mG mice from PBS, Losartan and DHB group were euthanized on day 3 post tumor inoculation, the implanted tumor harvested and fixed in pre-chilled 95% ethanol overnight at 4° C. before subjecting them to subsequent processing.
For metastatic melanoma studies, mice were intravenously injected via tail vein with 2×105 B16-F10 WT or B2m-cells. Mice were euthanized 14 days post-injection, lungs were then harvested, fixed in 4% Paraformaldehyde (PFA, Sigma Aldrich, catalog no. P6148) and collected for histological analysis. Blinded quantification of the metastatic lung foci was performed on macroscopic images and the size of metastatic foci was measured using ImageJ software.
Terminal subcutaneous tumors were excised with the skin and weighed before cutting in half. One half was placed in appropriate fixative while the other half was separated from the skin and chopped with scissors into ˜1 mm pieces. These were transferred to a 15 mL tube with 10 mL digest buffer (RPMI 1640 (Life Technologies, catalog no. 21870076), 200 U/mL Collagenase IV (Worthington Biochemical, Lakewood, NJ, USA)) and incubated for 2 hours at 37° C. with shaking. Following incubation, digested tumor was poured onto a 70 μM strainer placed in a 50 mL tube. The 15 mL tube was rinsed with ˜3 mL of 1×PBS/2% FCS/5 mM EDTA and added to the digested sample. The tumor was mashed with a plunger through the 70 μM strainer which was then rinsed ˜3 times with a small volume of 1×PBS/2% FCS/5 mM EDTA. The tumor-infiltrating CD45+ immune cells were isolated by magnetic-based positive selection using CD45 microbeads (Miltenyi Biotec, catalog no. 130-052-301) according to the manufacturer's protocol. The stained cells were passed through the LS columns (Miltenyi Biotec, catalog no. 130-042-401) placed between magnets for isolation of immune cells. The isolated CD45+ cells were centrifuged at 300 g for 5 minutes at 4° C. and resuspended in 2.4G2 blocking solution before flow cytometry staining.
For cytokine treatment, mice were injected with 100 ng/mouse of mIL-12 (Biolegend, catalog no. 577002), 2 μg/mouse of mIL-15 (Shenandoah Biotechnology, Warwick, PA, catalog no. 200-07) and 100 ng/mouse of mIL-18 (Shenandoah Biotechnology, catalog no. 200-83) in 200 μL of sterile 1×PBS. For the combination Poly(I:C) and cytokines, mice were injected with 200 μg/mouse of Poly(I:C) (Sigma-Aldrich, catalog no. P9582), 2 μg/mouse of mIL-15 (Shenandoah Biotechnology, catalog no. 200-07) and 100 ng/mouse of mIL-18 (Shenandoah Biotechnology, catalog no. 200-83) in 200 μL of sterile 1×PBS. Treatments were injected I.P. starting the day of the skin transplant (day 0), day 2 and day 4 and then injected S.C. at days 7, 10, 13, 16 and 19 post-transplant. Diphtheria toxin (DT, Sigma-Aldrich, catalog no. D0564) was injected I.P. at 500 ng/mouse the day before the transplant and 200 ng/mouse at days indicated in
Mice were I.P. injected with 500 μg/mouse of IgG isotype control (Southern Biotech, Birmingham, AL, USA, catalog no. 0107-01) or depleting anti-NK1.1 antibody (clone PK136, BioXcell, West Lebanon, NH, USA, catalog no. BE0036) two days before tumor cell injection and 250 μg/mouse every other day starting the day of the tumor cell injection. For depletion of NK or T cells (anti-CD4 antibody, clone GK1.5, BioXcell; anti-CD8a antibody, clone YTS 169.4, BioXcell) in a skin transplant setting, 500 μg/mouse of each depleting antibody were injected the day of the transplant and 250 μg at days indicated in
Prior to the experiment, blood from C57BL/6 WT and m157tg mice was collected, incubated overnight at 4° C., centrifuged and serum was isolated from the pellet. Spleens from C57BL/6 mice were harvested, mashed through a 70 μm filter and cells were resuspended in RBC lysis for two minutes. After washing with 1×PBS/2% FCS/5 mM EDTA, cells were counted and 5×106 splenocytes resuspended in 500 μL RPMI 10% FBS/1% P/S/20 mM Glutamine and 10% serum of either WT or m157tg mice then plated in a 24-well plate and incubated for 3 h at 37° C./5% CO2. Cells were then washed, re-plated and 200 μL of a mixture containing 1×105 WT-MEF or m157-MEF, mIL-12 (10 ng/mL, Biolegend, catalog no. 577002) or mIL-12/mIL-18 (12.5 ng/mL, Shenandoah Biotechnology, catalog no. 200-83) was added to the well. After incubation for another hour at 37° C./5% CO2, 1 μL of anti-CD107a-eF660 (clone 1D4B, eBioscience, catalog no. 50-1071-82), 1×Brefeldin A (Biolegend, catalog no. 420601) and 1×Monensin (Biolegend, catalog no. 420701) were added and cells were incubated for 6 hours. Then, the supernatant was removed, cells were washed and resuspended in 2.4G2 blocking solution before flow cytometry staining. Cells were assayed on a BD FACSCanto (BD Biosciences).
For in vitro experiments in the presence of ECM components, Collagen I (Rat tail, Sigma, catalog no. 08-115), collagen III (Human placenta, Sigma, catalog no. C4407), collagen IV (Mouse, Sigma, catalog no. C0543), Elastin (Mouse lung, Sigma, catalog no. E6402), Laminin (Mouse, Thermo Fisher, catalog no. 23017015) and Fibronectin (Rat plasma, Sigma, catalog no. F0635) were diluted in 1×PBS and coated at 20 μ ENREF 45 g/cm2 in a 24 well tissue culture plate for 30 minutes at RT with gentle shaking followed by overnight at 4° C. Spleens from Ncr1iCre, ROSAmT-mG, WT or Lair1−/− mice were harvested as described above. Then, 5×106 splenocytes resuspended in RPMI 10% FBS/1% P/S/20 mM Glutamine were mixed with 20 μg of respective ECM protein per well and were incubated at RT for 10 minutes. Splenocytes and ECM mix were plated onto the ECM-coated well and were incubated at 37° C./5% CO2 for 2 hours (short ECM exposure) or 17 hours (long ECM exposure). At the end of the incubation, 1×105 MEF (WT or m157) cells were added to each well to obtain a 50:1 splenocyte:MEF ratio. For cell signaling experiments, hydrogen peroxide (H2O2) at a final concentration of 2.5 mM or 1×105 MEF-m157 cells were added to each well except in “0 min” wells which were unstimulated and fixed immediately. Splenocytes were stimulated with 2.5 mM H2O2 for 2 min and 5 min or were co-cultured with MEF-m157 for 30 min and 60 min (signaling) or 7 hours (CD107a and IFN□) at 37° C./5% CO2. Splenocytes and MEF in co-culture were supplemented with mIL-12 (10 ng/mL, Biolegend, catalog no. 577002), mIL-15 (1 ng/mL, Shenandoah Biotechnology, catalog no. 200-07) and hIL-2 (Biolegend, catalog no. 589104; only long ECM exposure) as well as for CD107a/IFN experiments. 1 μL of anti-CD107a-BV421 (1D4B, Biolegend, catalog no. 121618), 1×Brefeldin A (Biolegend, catalog no. 420601) and 1×Monensin (Biolegend, catalog no. 420701) were added. Following the co-culture, cells were washed with 1×PBS/2% FCS/5 mM EDTA and the pellet was resuspended in 2.4G2 blocking solution before flow cytometry staining.
NK cells purified from spleens under sterile conditions were used for LEGENDplex experiments. Spleens from 8-9-week-old C57BL/6 female WT mice were harvested and mashed through a 70 μm filter. Cells were resuspended in RBC lysis for two minutes and washed with 1×PBS/2% FCS/5 mM EDTA. NK cells were enriched by negative selection using an NK cell isolation kit (Milteny Biotec, catalog no. 130-115-818) and magnetic LS columns (Milteny Biotec, catalog no. 130-042-401) according to the manufacturer's instructions. Enrichment of isolated NK cells was confirmed by flow cytometry and was 88-90% CD3−NK1.1+NKp46+ cells. Enriched NK cells were resuspended in RPMI 1640 media supplemented with 10% FBS and 1% P/S.
ECM protein coating on wells was performed as previously described. 1×105 enriched splenic NK cells was mixed with 20 μg of respective ECM protein per well in RPMI 1640/10% FBS/1% P/S/20 mM Glutamine and incubated at RT for 10 minutes. NK cells and ECM mix were plated onto the ECM-coated well and were incubated at 37° C./5% CO2 for 2 hours. At the end of the incubation, 1×105 MEF (WT or m157) cells were added to the respective wells to obtain a 1:1 NK:MEF ratio. NK cells and MEF were co-cultured at 37° C. 5% CO2 for 24 hours supplemented with mIL-12 (10 ng/mL, Biolegend, catalog no. 577002) and mIL-15 (1 ng/ml, Shenandoah Biotechnology, catalog no. 200-07-100 μg). The supernatant was collected at 12- and 24-hour intervals, centrifuged to remove cells/cell debris and subsequently frozen at −80° C. until further use. Quantification of murine cytokines and chemokines (CCL2, CCL5, CXCL9, CXCL10) in supernatants was performed with LEGENDplex kits (Biolegend, catalog nos. 740451, 740622) according to manufacturer's instructions. In brief, a mix of capture beads with distinct size and fluorescence was incubated with undiluted S/N or standard dilutions for 2 hours at RT with constant shaking. Beads were washed, a biotinylated detection antibody was added and the plate was incubated for 1 hour at RT with constant shaking. The streptavidin-PE conjugate was added and further incubated for 30 minutes at RT on a shaker. Beads were washed and subsequently acquired on a BD LSRFortessa X-20 flow cytometer. Analysis and quantification of the results were done using LEGENDplex data analysis software (BioLegend). Quantification was reported as pg/ml for CCL2, CCL5, CXCL10 and as MFI for CXCL9.
Skin grafts were collected and incubated in 4% PFA/25% sucrose (Fisher Scientific, Hampton, NH, USA, catalog no. S5-3) solution at 4° C. for 16 hours. The next day, slices were equilibrated for 4 h in 50% sucrose solution, embedded in OCT (Fisher Scientific, catalog no. 23-730-571), snap frozen and stored at −80° C. For paraffin embedding, lungs and tumor were collected and fixed in 4% PFA overnight at 4° C. Subsequently, tissues were dehydrated in ethanol, processed and embedded in paraffin according to standard histology processes. For paraffin embedding of Ncr1icre RosamT-mG a different fixation protocol was used to maintain the NKp46-GFP and Tdt-Tomato-ROSA fluorescence. Tumors from Ncr1icre, RosamT-mG mice were collected and fixed in pre-chilled 95% ethanol overnight at 4° C. Tissues were subsequently dehydrated in 100% ethanol, cleared in 100% xylene and embedded in paraffin according to standard histology processes. For IF staining of frozen tissue, 7 μm sections of skin were cut on a cryostat, re-hydrated in three washes of 1×Tris-buffered-saline (TBS) for 2 minutes each followed by blocking of nonspecific protein with 5% bovine serum albumin (BSA, Fisher Scientific, catalog no. BP1600) and 5% goat serum (Sigma-Aldrich, catalog no. G9023) in 1×TBS. Sections were stained overnight at 4° C. with primary antibodies (Table 2). The following day, slides were washed as above and incubated for two hours at RT with secondary antibodies conjugated to fluorochromes (Table 2). After washing as above, sections were incubated with a 1:4000 dilution of DAPI (Invitrogen, Carlsbad, CA, catalog no. D3571) in 1×TBS for 5 minutes at RT, washed, air-dried and coverslips mounted with Prolong Gold Antifade Reagent (Invitrogen, catalog no. P36930). Five or six randomly selected fields of view at 200× total magnification were obtained for each section using a Zeiss Axio Scan (Zeiss, Oberkochen, Germany). Blinded manual counting of NKp46+, CD3+, CD4+ and CD8+ T cells were performed using ZEN Blue Software (Zeiss, Oberkochen, Germany). Automated counting was performed for CD11b, F4/80 and Arginasel stains in the whole tumor region of the scanned slides by Halo3.0 software (Indica Labs, Albuquerque, USA). For IF of paraffin-embedded tissues, 5 μm sections were rehydrated and permeabilized with 1×PBS supplemented with 0.2% Triton X-100 (Thermo Fisher Scientific, catalog no. BP151) for 5 minutes. Antigen retrieval was then performed using a Cuisinart pressure cooker for 20 minutes at high pressure in antigen unmasking solution (Vector Laboratories, Burligame, CA, catalog no. H-3300). Slides were then washed three times for three minutes each in 1×PBS supplemented with 0.1% Tween 20 (Sigma-Aldrich, catalog no. P1379). Sections were blocked, stained (Table 2) and mounted as described above. Elastin fiber staining was performed using Verhoeff Van Gienson Elastin Stain kit (ab 150667, Abcam) following the manufacturer's protocol. For collagen staining, Masson's Trichrome staining (Polysciences, Warrington, PA, catalog no. 25088) was performed following the instructions of the manufacturer. Whole slide imaging was performed using a NanoZoomer S60 Digital slide scanner (Hamamatsu, Japan) or Axio Scan.Z1 (Zeiss, Germany) and analyzed with NDP-view2 software (Hamamatsu) or ZEN Blue Software (Zeiss, Oberkochen, Germany), respectively.
B2M gene alterations and expression in solid cancer and leukemia samples in TCGA and other public databases were obtained and analyzed through cBioPortal for Cancer Genomics at cbioportal.org.
Graphs show mean values±standard deviation (SD). The numbers of mice per group used in each experiment are annotated in the corresponding figure legend as n. Graphs and statistical analysis were performed using GraphPad Prism 8 (La Jolla, CA, USA) and RStudio. All tumor quantifications were performed blindly. Two-tailed Fisher's exact test was used to compare skin graft rejection grades and lymph node metastatic load among groups. Two-way ANOVA with Sidak's multiple comparison test was used to compare tumor growth over time between different groups. Comparisons of survival were performed with the Log-rank test. Two-tailed Mann-Whitney U test was used for all the other comparisons. A P value of less than 0.05 was considered significant.
In order to assess the requirements for NK cell cytotoxicity in solid organs, we developed a syngeneic skin transplant model. Ear skin from donor mice on an albino B6 background was transplanted onto the back of B6 recipients. WT and m157tg mice with or without the deletion of beta-2 microglobulin (B2m) gene were used as donors. Ncr1iCre, ROSAmT-mG reporter mice expressing green fluorescent protein (GFP) in NKp46+ NK cells and TdTomato in all other cells were used as recipients (
Next, we examined the strength of m157-Ly49H interaction in NK cells that infiltrated the skin grafts. Recipient-derived CD49b+ cNK and CD49a+ trNK cells were found in m157-expressing skin grafts, which expressed Ly49H and exhibited m157-induced Ly49H downregulation compared with Ly49H− and Ly49H− cNK and Ly49H− trNK cells in the recipient's liver (27). No significant alteration in NKG2D, CD48, DNAM1, NKG2A, Ly49A and Ly49C/I expression on the surface of CD49b+ cNK and CD49b″CD49a double-positive NK (dpNK) cells in m157tg, B2m−/− skin graft was detected. Although a large number of NKp46-GFP+ cNK and trNK cells infiltrated m157-expressing skin grafts, these cells did not upregulate IFNγ, TNFα or granzyme B expression. NK cells isolated from blood, spleen and lymph nodes of WT recipients transplanted with m157tg skin did not downregulate Ly49H expression in contrast to those of m157tg mice, ruling out systemic NK cell hyporesponsiveness in recipient animals (27). In addition, WT NK cells were fully responsive to m157 stimulation in the presence of serum from m157tg mice in vitro, ruling out the possibility that a soluble form of m157 blocked NK cell activation in m157-expressing skin grafts. NK cells can become hyporesponsive upon chronic exposure to the loss of MHC-I (28, 29). To investigate this concept in our transplant system, we tested the ability of m157tg, B2m−/− skin graft-infiltrating NK cells at day 10 post transplantation to degranulate and produce IFNγ in response to ex vivo stimulation. Compared with splenic cNK cells, skin graft-derived cNK cells had comparable expression levels of MHC-I specific inhibitory receptors and were not found to be degranulating at baseline. However, upon ex vivo co-culture, Ly49H+ cNK cells from m157tg, B2m−/− skin graft showed robust degranulation and IFNγ secretion compared with splenic Ly49H− cNK cells in control IgG-coated, aNK1.1 antibody-coated plates as well as upon phorbol myristate acetate plus ionomycin (PMA/Ion) stimulation. We did not observe any increase in degranulation and IFNγ secretion by Ly49H cNK cells in the skin graft versus spleen except for a minor increase in degranulation of skin Ly49H− NK cells in IgG-coated plate. These findings demonstrate that Ly49H″ cNK cells from m157tg, B2m−/− skin graft are not hyporesponsive and are fully capable of activation and degranulation outside of the skin.
To investigate the timing of inflammation in m157-expressing skin grafts, we compared m157 g grafts to skin grafts expressing a model T cell antigen, Ovalbumin (OVA). WT B6 mice were transplanted with m157tg, m157tg, B2m−/−, mOVAtg and mOVAtg, B2m−/− skin grafts and monitored. As expected, mOVAtg donor skin grafts were mostly rejected by day 20 post-transplant while the absence of MHC-I inhibited the rejection of mOVAtg, B2m grafts (FIG. 1G). The paradoxical increase in inflammation in m157tg, B2m−/− compared with m157tg grafts supported the dominant role of NK rather than T cells in initiating the immune response in m157-expressing grafts. However, we noticed that inflammation in m157tg,B2m−/− skin grafts developed after day 12 post-transplant, along a similar adaptive immune response timeline to mOVAtg grafts (
To overcome the apparent inhibition of cytotoxicity experienced by NK cells in m157-expressing skin grafts, we treated the recipient mice with IL-15, IL-18 and a TLR3 agonist, poly(I:C), or IL-12, IL-15 and IL-18 (
To determine whether NKp46-GFP+ NK cells in m157tg, B2m−/− skin grafts were recruited from circulation or the surrounding recipient skin, we performed a parabiosis experiment. Ncr1iCre, ROSAmT-mG mice were partnered with WT mice and their circulatory systems were allowed to conjoin for 20 days before m1571 g, B2m−/− skin was transplanted onto the WT parabiont. At 20 days following skin transplantation, NKp46-GFP+ NK cells were found in the spleen and the skin graft, the majority of which were negative for anti-CD45 antibody injected I.V. NKp46-GFP+ NK cells migrated into the dermis and epidermis of m157tg,B2m−/− skin followed by a large T cell infiltrate into the skin grafts. To directly test recruitment from circulation, we sorted splenic NKp46-GFP CD49b+CD49a− cNK cells from Ncr1iCre, ROSAmT-mG mice and injected them I.V. into WT mice one day following m157tg, B2m−/− skin transplantation. After 20 days, NKp46-GFP+ NK cells were readily identified circulating in the recipient liver and a few NKp46-GFP+ NK cells were detected in the skin grafts, which had exited the circulation. Together, these data demonstrate that NK cells in the donor graft are recruited from the circulation and migrate into the dermis and epidermis where they likely adopt tissue-resident cell properties.
Next, we took genetic approaches to block trNK cell development, and thereby augment the cytotoxic function of cNK cells in the skin. In a cancer setting, enhanced NK cell cytotoxicity is accomplished through the elimination of the immunosuppressive signal from TGFβ (37), which converts cNK to trNK cells (13). WT and Ncr1iCre, Tgfbr2fl/fl mice were transplanted with m157tg, B2m−/− skin grafts; however, no rejection was observed at day 20 post-transplant. Likewise, the deletion of transcription factor Hobit, which is required for the development of liver trNK cells (38), in recipient mice did not impact m15718, B2m−/− skin grafts after 20 days. Quantification of the frequency of cNK and trNK cells in the recipient liver and skin grafts revealed increased cNK and decreased trNK cells in m157tg, B2m−/− skin grafts in Ncr1iCre, Tgfbr2fl/fl and Hobit−/− compared with WT recipients. Therefore, the signal(s) in the skin that restrict NK cell cytotoxicity are not superseded by exogenous NK cell-stimulating factors, restriction of immunosuppressive TGFβ signaling or prevention of cNK to trNK cell conversion.
Our previous attempts to induce rejection of skin grafts through activation of the Ly49H pathway together with loss of MHC-I failed in a syngeneic transplant system. In order to preserve CD8+ T cell functionality while removing NK cell inhibition through the absence of self MHC-I, we generated Ncr1iCre, ROSAmT-mG recipient mice that were first-generation (F1) progeny of B6×BALB/c parents and transplanted them with WT and m157tg B6 skin grafts. In this setting, CD8+ T cells were still functional through matched MHC-I signaling while NK cells licensed by BALB/c MHC-I lost MHC-I-mediated inhibition against B6 donor skin cells. Although WT skin grafts remained intact in F1 recipients, the majority of m157tg skin grafts were rejected by day 20. The remainder of m157tg grafts were fully rejected by day 60 post-transplant. m157tg skin graft size was significantly reduced compared with WT grafts starting from day 16 post-transplant, similar to the timeline of mOVAtg skin graft rejection in WT B6 mice. Thus, an NK cell-specific activating ligand in the context of missing-self in F1 recipients induces skin graft rejection; however, this rejection does not initiate within a 48-72-hour window expected of NK cell direct cytotoxicity. Since BALB/c mice do not express Ly49H, the proportion of Ly49H-expressing cNK cells in the B6×BALB/c F1 mice was reduced compared with B6 mice. However, similar to B6 recipient mice (
Significant increases in the number of NKp46-GFP+ NK, CD4+ T and CD8 T cells were found in m157tg skin grafts at day 10 post-transplant. Quantifying the immune cell infiltrates over time revealed that significant numbers of NKp46-GFP+ NK cells appeared in m157tg skin grafts at day 5 post-transplant while CD4+ T and CD8+ T cells infiltrated m157tg skin grafts with a delay (
To define the function of NK cells that infiltrated m157-expressing skin grafts, we examined the transcriptional profiles of cNK cells in the circulation (spleen and blood), cNK cells recently emigrated from the circulation into m157-expressing skin grafts, dpNK and trNK cells in the skin grafts and trNK cells in the recipient skin. This strategy enabled us to identify pathways that were up or downregulated in cNK cells early during their entry into the skin microenvironment. Strikingly, we discovered a dramatic switch in cNK cells' profile as they entered the skin grafts, which was highlighted by downregulation of cytotoxicity-related genes and upregulation of chemokines and inflammatory cytokines. Ccl1, Ccl2, Cxcl2, Cxcl9 and Cxcl10 were significantly upregulated in cNK cells in the skin graft compared to cNK cells in circulation. Likewise, Il1a, Il1b and Tnf were upregulated in donor skin cNK cells. No upregulation was seen in Ifng, Gzma or Gzmb expression, and Prf1 (perforin), Klrk1, Ccl5, Eomes and Thx21 (T-bet) were significantly downregulated in donor skin cNK cells compared with their baseline expression in circulating cNK cells. Based on RNA-Seq data and confirmed by flow cytometry, we identified TGFβRII, IL-4Rα, CCR7 and TIGIT as novel markers to distinguish cNK cells in the skin from circulating cNK cells. Together, these data demonstrate that upon entry into the skin, cNK cells undergo a drastic change in their function, which involves downregulating their cytotoxic program while boosting their ability to provide ‘help’ to neighboring immune cell populations through the production of chemokines and inflammatory cytokines.
Considering the significant upregulation of Tigit, Pdcd1 and Ctla4 in cNK cells entering the skin grafts, we examined the impact of immune checkpoint blockade on the rejection of m157-expressing skin grafts. PD-1, TIGIT or TIGIT/PD-1/CTLA4 triple antibody blockade did not result in rejection of m157tg or m157tg,B2m−/− skin grafts in WT B6 recipients. Further, TIGIT/PD-1/CTLA4 triple antibody blockade did not accelerate the rejection of m157tg skin grafts in F1 recipients, indicating the persistent lack of NK cell direct cytotoxicity in the skin grafts.
To identify the mediator(s) of NK cell functional switch in the skin, we examined the components of the skin microenvironment that interacted with NK cells as soon as they entered the skin grafts at day 5 post-transplant. NK cells infiltrated broadly into m157tg dermal ECM, surrounded by collagen and to a lesser extent elastin. In addition, occasional NK cells were detected contacting dermal fibroblasts at day 5 post-transplant. To elucidate the impact of ECM proteins and fibroblasts on cNK cell cytotoxicity, we examined splenic cNK cell degranulation and cytokine production in co-culture assays. WT mouse embryonic fibroblasts (WT-MEF) did not block Ly49H+ cNK cell degranulation or IFNγ production in response to IL-12 and IL-15 stimulation (
To gain a greater insight into the underlying molecular mechanisms linking the ECM to changed NK cell functionalities, we investigated the signaling pathways that were associated with the switch in cNK cells' profile from cytotoxicity to an inflammatory response. cNK cells entering the skin downregulated PI3K-AKT pathway while upregulating NFκB, STAT3 and STAT5 signaling pathways compared with circulating cNK cells (
Considering the essential role of ECM proteins in skin homeostasis and wound healing, our attempts to remove or degrade ECM proteins in the skin transplantation system led to the failure of skin engraftment. Thus, we examined the role of ECM proteins in suppressing NK cell cytotoxicity against cancer cells in the skin. We subcutaneously (S.C.) injected B2m−/− B16-F10 melanoma cells (B16) into the flanks of WT mice and treated them with control IgG or anti-NK1.1 antibody to determine if NK cells inhibited the growth of MHC-I-deficient tumors in the skin (
To translate our fundamental findings to human cancers, we analyzed the TCGA and other publicly available datasets for the frequency of B2M gene alteration and mutations in multiple types of solid cancers and leukemias. While B2M gene mutations and deletion were found across solid cancer types including those with low mutational burden, leukemias did not carry any aberrations in the B2M gene (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/188,122, filed on May 13, 2021, and 63/275,517, filed on Nov. 4, 2021. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/072319 | 5/13/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63275517 | Nov 2021 | US | |
| 63188122 | May 2021 | US |
