Patent Application
20240067956
A Sequence Listing in ASCII text format, entitled 151077-00044_ST25.txt, 555 bytes in size, generated on Jan. 12, 2022, and filed via EFS-Web, is provided with this application. The Sequence Listing is incorporated herein by reference into the specification for its disclosures.
The present invention relates to methods and compositions related to tracking uptake and distribution of molecules and/or molecular structures, such as antigens and antigenic molecules and/or molecular structures, resulting from, for example, an infection or vaccination.
Depending on the route of infection, vaccination mode, and ability of antigens to traffic, different dendritic cell (DC) subsets are required to initiate T cell priming. Upon subcutaneous immunization, small soluble proteins and virus particles pass through the lymphatics to the lymph node (LN), where LN-resident DCs acquire and present antigen1,2. For larger antigens and/or pathogens that are too large to pass through the lymphatic capillaries, dermal DCs migrate to the LN for presentation of processed antigens to naïve T cells1,2,3,4. Most adaptive immune responses require antigen processing and presentation by conventional dendritic cells in either the draining lymph node or at the site of infection or vaccination (migratory cutaneous or dermal DCs)5.
Previous studies have shown that viral antigens persist in the lymph node beyond the time frame of infectious virus6,7,8,9,10,11. Lymphatic endothelial cells (LEC) were recently found to store antigens from viral infection and vaccination12,13,14. Using a vaccine formulation that elicits robust cell-mediated immunity comprised of antigen, a Toll-like receptor (TLR) agonist, and an agonistic aCD40 antibody (TLR/αCD40 vaccination) or viral infection15,16,17,18,19,20,21,22,23,24,25,26, it was discovered that antigens were durably retained in the lymph node12,13,14. Antigen storage was dependent on the presence of a TLR agonist (e.g. polyI:C alone (TLR3/MDA5/RIGI or Pam3cys (TLR1/2)+αCD40), but also occurred with antigen conjugated to a TLR agonist (e.g. 3M019 (TLR7))14. This process has been described as “antigen archiving,” and it has been shown that this process is important to poise memory T cells for future antigenic encounters14.
Prior to these studies, the only non-hematopoietic cell type thought to retain antigens were follicular dendritic cells, which harbor antigens in antigen-antibody complexes for extended periods of time and for the benefit of B cell memory11,27. Fibroblasts and non-endothelial stromal cells comprise a large portion of the lymph node stroma and are capable of presenting peripheral tissue antigens, but their capacity to acquire and present foreign antigens is not yet well understood28,29,30. Initial studies were unable to detect antigen archiving by blood endothelial cells or fibroblasts12,13. While LECs have been shown to present antigens in the absence of inflammation to induce T cell tolerance31,32,33,34,35,36,37,38, it has been shown that presentation of archived antigen occurs only after exchange of the archived antigen from an LEC to a migratory DC; changing the stimulus from tolerizing to immunostimulatory12,13. Soluble antigens are exchanged via two distinct mechanisms: (i) direct exchange between LECs and migratory DCs and (ii) LEC death. Antigen transfer from LECs to both migratory conventional (c)DC1s and cDC2s is required for archived antigen presentation to antigen-specific memory T cells12,13. After viral infection, archived antigen is transferred to Batf3 dependent migratory DCs as a result of LEC death during lymph node contraction12.
Limitations of current approaches have precluded sensitive and quantitative measures of antigen levels across cell types, providing only a glimpse of the cell types and molecular mechanisms that control antigen acquisition, processing and retention in the lymph node. Studies of antigen in the lymph node and peripheral tissues have mainly relied on antigen-fluorophore conjugates or indirect measurement of antigen uptake and presentation2,6,7,11,12,14,39, which defined antigen acquisition by specific DC subsets and trafficking of antigens using live imaging2. However, antigen archiving has been difficult to study because antigen-fluorophore conjugates suffer from low microscopic detection sensitivity, yielding weak signals that diminish over time. Moreover, detection of antigen in the lymph node and other tissues has relied on flow cytometric analysis using cell surface markers, restricting analysis to specific cell types. Furthermore, shortcomings related to the use of antigen/antibody-DNA conjugates in tracking antigens include the rapid degradation of these conjugates in vivo. Thus, in order to address these limitations in current approaches to detecting antigen acquisition and/or uptake, improved approaches to molecular tracking, such as in tracking antigen molecules throughout tissue-specific cell types in vivo, are needed.
The present inventive concept relates to methods and compositions of molecular tracking in individual cells in vivo using single-cell mRNA-sequencing. According to an aspect of the inventive concept, provided is a molecular conjugate including: a molecule or molecular structure of interest; and a nucleic acid linked to the molecule or molecular structure of interest, wherein the nucleic acid includes a nucleic acid amplification primer binding sequence, an identifier sequence, and a capture sequence, and wherein the nucleic acid includes a phophorothioate modification at every nucleotide linkage.
According to another aspect of the inventive concept, provided is a method of tracking acquisition of a molecule or molecular structure of interest in cells including: exposing a subject to a molecular conjugate of the inventive concept; performing single-cell nucleic acid sequencing on cells of interest derived and/or isolated from the subject; and analyzing data acquired from the single-cell nucleic acid sequencing for a presence of the identifier sequence, wherein the presence of the barcode identifier sequence is indicative of acquisition of the molecule or molecular structure of interest by a particular cell or cells.
According to another aspect of the inventive concept, provided is a method of tracking distribution or location of a molecule or a molecular structure of interest in a subject including: exposing a subject to the molecular conjugate of the inventive concept; performing single-cell nucleic acid sequencing on cells of interest derived and/or isolated from the subject; and analyzing data acquired from the single-cell nucleic acid sequencing for a presence of the barcode identifier sequence, wherein the presence of the barcode identifier sequence is indicative of acquisition of the molecule or molecular structure of interest by a particular cell or cells in the subject, and where the molecule or molecular structure of interest is distributed or located in the subject.
According to another aspect off the inventive concept, provided is a method of molecular tracking including: exposing a subject to the molecular conjugate of the inventive concept; performing single-cell nucleic acid sequencing on cells of interest derived and/or isolated from the subject; and analyzing data acquired from the single-cell nucleic acid sequencing for a presence of the barcode identifier sequence, wherein the presence of the barcode identifier sequence is indicative of acquisition of the molecule of interest by a particular cell or cells.
According to another aspect of the inventive concept, provided is a method of tracking antigen archiving including: exposing a subject to a molecular conjugate including an antigen of interest linked to a nucleic acid, wherein the nucleic acid includes a nucleic acid amplification primer binding sequence, a barcode identifier sequence, and a capture sequence, and wherein the nucleic acid includes a phophorothioate modification at every nucleotide linkage; performing single-cell nucleic acid sequencing on cells of interest derived and/or isolated from the subject; and analyzing data acquired from the single-cell nucleic acid sequencing for a presence of the barcode identifier sequence, wherein the presence of the barcode identifier sequence is indicative of acquisition of the antigen of interest by a particular cell or cells.
For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The definitions contained in this specification are provided for clarity in describing the components, compositions, and methods herein and are not intended to limit the claimed aspects and embodiments of the inventive concept.
Articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element. The term “and/or” includes any and all combinations of one, or more, of the associated listed items and may be abbreviated as “/”.
The term “comprise,” as used herein, in addition to its regular meaning, may also include, and, in some embodiments, may specifically refer to the expressions “consist essentially of” and/or “consist of.” Thus, the expression “comprise” can also refer to, in some embodiments, the specifically listed elements of that which is claimed and does not include further elements, as well as embodiments in which the specifically listed elements of that which is claimed may and/or does encompass further elements, or embodiments in which the specifically listed elements of that which is claimed may encompass further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed. For example, that which is claimed, such as a method, kit, system, etc. “comprising” listed elements also encompasses, for example, a composition, method, kit, etc. “consisting of,” i.e., wherein that which is claimed does not include further elements, and a composition, method, kit, etc. “consisting essentially of,” i.e., wherein that which is claimed may include further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed.
The term “molecular conjugate” refers to a construct including a molecule and/or molecular structure of interest linked (covalently, non-covalently, or otherwise as described herein) to a nucleic acid. the nucleic acid may include several functional elements, such as: a binding sequence for a nucleic acid amplification primer, also referred to, in some embodiments, as a PCR handle; an identifier sequence, for example, a barcode identifier sequence that specifically identifies that linked/attached molecule and/or molecular structure of interest; and a capture sequence that includes a sequence that is complementary to, for example, a binding/hybridizing sequence included on a gel bead, such as is available from 10× Genomics, but not limited thereto. The nucleic acid may further include a unique molecular identifier (UMI) locate, for example, either 3′ or 5′ to the identifier/barcode identifier sequence.
A single strand of a nucleic acid typically includes a 5′ (5-prime) end and a 3′ (3-prime) end. The terms 5′ and 3′ therefore refer to a relative position on a single strand of a nucleic acid. Accordingly, the relative position of certain elements or sequences of a nucleic acid (e.g., a nucleic acid amplification primer binding sequence/amplification or PCR handle, an identifier sequence/barcode and a capture sequence) can be specified in a sequential order from 5′ to 3′, or alternatively from 3′ to 5′. It will be appreciated that generally, elements/sequences of a nucleic acid are referred to from 5′ to 3′, unless otherwise specified. For example, a nucleic acid may include, from 5′ to 3′, a nucleic acid amplification primer binding sequence, an identifier sequence/barcode and a capture sequence, and may be represented as: 5′-PCR handle-barcode-capture sequence-3′. In the above example, the barcode and the capture sequence may be referred to as being 3′ of the PCR handle. Also, in the above example, the handle and the barcode may be referred to as being 5′ of the capture sequence. Further, the position of the handle in the above example may also be referred to as adjacent to the barcode. Similarly, the barcode may be referred to as flanked by the handle and the anchor. Accordingly, one of skill in the art would know what is meant by the positional terms 3′ and 5′. Such positional language, as used herein, unless explicitly indicated otherwise, does not imply that additional nucleic acid sequences are not interposed between the reference elements. For example, in the above example, additional sequences (e.g., a UMI) may be present between the PCR handle and the barcode, or the barcode and the capture sequence.
The terms “linked to” or “attached to” as used herein to describe the interaction between the components of the conjugates include covalent linkages/attachments or a variety of non-covalent types of linkages/attachments. Chemistries useful in assembling the constructs described herein include, but are not limited to, thiol-maleimide, thiol-haloacetate, amine-NHS, amine-isothiocyanate, and “click” chemistries, such as, but not limited to, Copper(I)-catalyzed azide-alkyne cycloadditions (CuAAC), strain-promoted azide-alkyne cycloadditions (SPAAC) and inverse electron-demand Diels-Alder (IEDDA) additions, such as a methyltetrazine-trans-cyclooctene addition. In some embodiments, each nucleic acid is linked to the molecule or molecular structure of interest by an irreversible covalent link. In other embodiments, each nucleic acid is linked to the molecule or molecular structure of interest by a cleavable covalent link, for example a disulfide link or a photocleavable linker.
A “molecule of interest” may be any naturally occurring or synthetic biological or chemical molecule. In some embodiments, the molecule of interest may be a peptide or protein. In some embodiments, the molecule of interest may be any antigen or antigenic molecule. A “molecular structure of interest” may refer to a feature and/or characteristic of a molecule of interest. For example, in some embodiments, the molecular structure may include an amino acid or nucleic acid sequence, or the molecular structure may include a sugar and/or glycan chain. In some embodiments, the molecular structure may include a primary sequence, or a secondary or tertiary structure of a peptide, protein, or nucleic acid, or a secondary of tertiary structure of a sugar/glycan chain. In some embodiments, the molecule/molecular structure of interest may be an active agent, such as a component of a therapeutic/pharmaceutical composition/formulation, such as a vaccine.
In general, the nucleic acid/oligonucleotide can be any length that accommodates the lengths of its functional components. In some embodiments, the nucleic acid/oligonucleotide is between about 20 and 100 nucleic acid bases/nucleotides in length. In some embodiments, the nucleic acid/oligonucleotide sequence is at least about 20, 30, 40, 50, 60, 70, 80, 90 or over 100 nucleic acid bases/nucleotides in length. In other embodiments, the nucleic acid/oligonucleotide is about 200 to about 400 monomeric components, e.g., nucleic acid bases/nucleotides in length. In some embodiments, the nucleic acid is generally made up of deoxyribonucleic acids (DNA). In some embodiments, the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid, or a part/portion thereof, includes modified DNA bases. Modification of DNA bases are known in the art and can include chemically modified bases including labels. In other embodiments, the nucleic acid, or a part/portion thereof, includes ribonucleic acid (RNA) sequences or modified ribonucleotide bases. Modification of RNA bases are known in the art and can include chemically modified bases including labels. In still other embodiments, different portions of nucleic acid/oligonucleotide sequence can include DNA and RNA, modified bases/nucleobases, or modified nucleic acid base/nucleotide connections, such as, for example, phosphorothioate DNA, peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In some embodiments, the nucleic acid/oligonucleotide includes phosphorothioate modifications, for example, such as phosphorothioate DNA, at some, most, or all nucleic acid base/nucleotide connections/linkages of the nucleic acid/oligonucleotide.
The term “amplification handle,” “amplification sequence” or, in some embodiments, “PCR handle” refers to a functional component of the nucleic acid/oligonucleotide sequence which itself is an oligonucleotide or polynucleotide sequence that provides a binding/annealing/hybridizing site and/or sequence for amplification of the nucleic acid/oligonucleotide sequence by, for example, a nucleic acid amplification primer. The amplification or PCR handle can be formed of DNA, RNA, phosphorothioate DNA, PNA, LNA, modified bases or combinations of these bases, or polyamides, etc. In some embodiments, the amplification or PCR handle is about 10 nucleic acid bases/nucleotides in length. In other embodiments, the amplification or PCR handle is at least about 5 to 100 nucleic acid bases/nucleotides in length. Thus in various embodiments, the amplification or PCR handle is formed of a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 nucleic acid bases/nucleotides. In some embodiments, when present in multiple nucleic acid/oligonucleotide sequences, the amplification or PCR handle can be the same or different, depending upon the techniques intended to be used for amplification. In some embodiments, the amplification or PCR handle can be a generic sequence suitable as an annealing site for a variety of amplification technologies. Amplification technologies include, but are not limited to, DNA-polymerase based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) and polymerization by any number of DNA polymerases (for example, T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase) and RNA-polymerase based amplification systems (such as T7-, T3-, and SP6-RNA-polymerase amplification), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase dependant amplification (HDA), ramification amplification method and RNA-seq.
The term “barcode” describes a nucleic acid/oligonucleotide sequence, that when it is a functional element of the nucleic acid, is specific for a single molecular conjugate. The barcode can be formed of a defined sequence of DNA, RNA, modified bases or combinations of these bases, as defined above. In some embodiments, the barcode is about 2 to 4 monomeric nucleic acid bases/nucleotides in length. In other embodiments, the barcode is at least about 1 to about 100 nucleic acid bases/nucleotides in length. Thus in various embodiments, the barcode is formed of a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 nucleic acid bases/nucleotides in length. According to embodiments of the inventive concept, the barcode may be used in identifying uptake of a molecular conjugate or conjugates by a particular cell or cells.
The term “capture sequence” refers to a polynucleotide or oligonucleotide sequence, which is designed to hybridize to a complementary oligonucleotide sequence, e.g., an oligonucleotide, a primer, and/or the like. In some embodiments of the nucleic acid, a capture sequence is designed for the purpose of generating a double-stranded oligonucleotide sequence. In some embodiments, the capture sequence is positioned at the 3′ end of the nucleic acid. In other embodiments, the capture sequence is positioned at the 5′ end of the nucleic acid. In some embodiments, each capture sequence is specific for its intended complementary sequence. For example, in certain embodiments, the capture is configured to hybridize to a 3′ end of a complementary oligonucleotide such that the 3′ end of the complementary oligonucleotide acts as a primer that can generate a second complementary strand of the oligonucleotide in the presence of a polymerase. When used in the various methods described herein, a capture sequence may hybridize to a free complementary sequence or with a complementary sequence that is immobilized on a substrate. In some embodiments, the capture sequence can be formed of a sequence of, e.g., DNA, RNA, phosphorothioate DNA, PNA, LNA, modified bases or combinations of these bases, or polyamides, etc. In some embodiments, the capture sequence is about 3 to 15 nucleic acid bases/nucleotides in length. In other embodiments, the capture sequence can be at least about 3 to 100 nucleic acid bases/nucleotides in length. In some embodiments, an anchor comprises 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 nucleic acid bases/nucleotides in length. In various embodiments, an Anchor is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 nucleic acid bases/nucleotides in length.
A “complementary oligonucleotide, “complementary binding oligonucleotide,” “complementary hybridizing oligonucleotide,’ “complementary oligo,” “complementary binding oligo” or “complementary hybridizing oligo” may be, e.g., an oligonucleotide/polynucleotide including at least a sequence that is complementary to the capture sequence of the molecular conjugate. In some embodiments, the complementary oligo is not part of the molecular conjugate; rather it is any oligonucleotide/polynucleotide that is part of a construct-purification kit or an mRNA-sequencing kit. The term “complementary sequence” may refer to a sequence to which a capture sequence (or other nucleic acid, e.g., a primer or capture oligonucleotide) is intended to hybridize/bind to, often resulting in a hybridized double stranded complex. In the presence of a polymerase, a hybridized complex can, in some embodiments, be extended in a 3′ direction where a nucleic acid template is present. Accordingly, in certain embodiments, a sequence complementary to the capture sequence can hybridize to a capture sequence thereby providing a primer for amplification and/or to generate a double stranded sequence. In some embodiments, the complementary oligonucleotide/polynucleotide may include sequences that can be used as amplification/PCR handles and optionally include one or more UMIs and barcode sequences. In the methods of the inventive concept, the extension of the complementary oligonucleotide/polynucleotide, with its complementary sequence hybridized to the capture sequence copies the barcode, the UMI and the amplification/PCR handle from the molecular conjugate onto the capture polymer/oligonucleotide. According to embodiments of the inventive concept, the capture polymer/oligonucleotide and its complementary sequence can be formed of DNA, RNA, phosphorothioate DNA, PNA, LNA, modified bases or combinations of these bases, or of any other component as defined above. Depending upon the assay steps involved and the intended target, the complementary sequence can be unhindered or “free” in the biological sample. In one embodiment, the complementary oligonucleotide/polynucleotide includes a sequence that is a primer sequence designed to participate in amplifying the molecular conjugate oligonucleotide sequence. In other embodiments, the complementary oligonucleotide/polynucleotide is immobilized on a substrate. Similar to the capture sequence, each complementary sequence can be at least about 3 to about 100 nucleic acid bases/nucleotides in length. Thus in various embodiments, the complementary sequence is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 nucleic acid bases/nucleotides in length. according to embodiments of the inventive concept, the complementary sequence may be any oligonucleotide sequence that may be envisioned by one of skill in the art, provided that it can hybridize to its intended capture sequence.
The term “immobilized” can refer to an oligonucleotide/polynucleotide including, for example, a complementary sequence as described herein, that is attached to a solid substrate resulting in reduction or loss of mobility through, for example, physical adsorption through charge-charge interaction or hydrophobic interaction, covalent bonding, Streptavidin-Biotin interaction, or affinity coupling.
The term “substrate” can refer to a microparticle/bead, a microfluidics microparticle/bead, a slide, a multi-well plate or a chip. The substrates are conventional and can be glass, plastic or of any conventional materials suitable for the particular assay or diagnostic protocols. In exemplary embodiments, substrates include gel beads, for example, gel beads-in-emulsion (GEMs) such as those that are commercially available from 10× Genomics, and the like.
The compositions and formulations used in methods of the inventive concept as described herein include molecular conjugates that include components selected based on the purpose of the assay/method, and the protocols used for the assay/method. The method used may dictate the selection and compositions of the various components described above which make up the composition. It will be appreciated that the following description is not exhaustive, and one of skill in the art can design many different compositions based on the teachings provided herein. The composition may also include the molecular conjugates in a suitable carrier or excipient. The elements of each composition will depend upon the assay that may be employed.
In some embodiments, a molecular conjugate may include a molecule or molecular structure of interest attached to or conjugated with a nucleic acid by, for example, a linker. The nucleic acid may be an oligonucleotide sequence including: a hybridizing/binding sequence for a nucleic acid amplification primer, i.e., an amplification handle and/or PCR handle; an identifier sequence, such as a barcode, that specifically identifies the molecule or molecular structure of interest; and a capture sequence for hybridizing/binding to a complementary oligonucleotide sequence to the capture sequence. The nucleic acid may, optionally, further include a UMI, which may by positioned adjacent to the identifier sequence/barcode, at either the 5′ or 3′ end of the identifier sequence/barcode.
The nature of the molecule or molecular structure of interest is not particularly limited, and as such may be any molecule or molecular structure that one of skill in the art may wish to track/monitor uptake of the molecule/molecular structure by a cell or cells that may be individual cells or part of a higher organism. Such tracking/monitoring may be tracking/monitoring of uptake of the molecule/molecular structure in a cell, cells, organs, and/or tissues of a subject. In some embodiments, for example, the molecule or molecular structure of interest may be one that behaves as an antigen or may have antigenic properties, such as a toxin or foreign substance/organism, which triggers an immune and/or inflammatory response in a subject. In some embodiments, the molecule or molecular structure of interest may be, for example, a therapeutic for promoting an immune response against a pathogen. In some embodiments, the molecule or molecular structure of interest may be a polypeptide/protein or a sugar/glycan/peptidoglycan, or a structure on a polypeptide/protein or structure on a sugar/glycan/peptidoglycan. In some embodiments, the molecule or molecular structure may be, for example, a polypeptide and/or protein of bacterial origin or nature. In some embodiments, the molecule or molecular structure may be a polypeptide and/or protein of viral origin or nature, for example, a polypeptide and/or protein from an influenza virus, or a polypeptide and/or protein from a coronavirus, such as SARS coronavirus-2 (SARS-CoV2). In other embodiments, the molecule or molecular structure of interest is nonimmunogenic, i.e., does not trigger an immune and/or inflammatory response against the molecule or molecular structure of interest, such as a therapeutic for treating a disorder, such as a viral infection. In some embodiments, the molecule or molecular structure is an active agent/part of an active agent that is a component in and/or an ingredient included in a vaccine.
Many types of molecules or molecular structures of interest, amplification primer binding sequences/PCR handles, and identifier sequences/barcodes can be used to generate a wide variety of molecular conjugates/compositions as described herein without departing from the scope of the inventive concept.
Kits containing the molecular conjugates/compositions of the inventive concept are also provided. Such kits will contain one or more molecular conjugates/compositions, one or more preservatives, stabilizers, or buffers, and such suitable assay and amplification reagents depending upon the amplification and analysis methods and protocols with which the molecular conjugate/composition will be used. Still other components in a kit include optional reagents for cleavage of the linker, a wash buffer, a blocking solution, a lysis buffer, and an encapsulation solution, detectable labels, immobilization substrates, optional substrates for enzymatic labels, as well as other laboratory items and instructions for use.
The molecular conjugates/compositions, and kits including the molecular conjugates/compositions of the inventive concept described herein may be used in a variety of methods by employing any number of assays/methods for detecting the molecular conjugate/composition in general.
In some embodiments, methods of the inventive concept include methods of tracking a molecule or molecular structure of interest, i.e., a method of molecular tracking, in a subject. In some embodiments, methods of tracking include tracking acquisition and/or uptake of the molecule or molecular structure of interest in the subject. In some embodiments, methods of tracking include tracking distribution or location of the molecule or molecular structure of interest in the subject. In some embodiments, methods of tracking include methods of tracking antigen archiving in the subject.
Methods of tracking, according to embodiments of the inventive concept, may include use of molecular conjugates/compositions as described herein to determine the presence, amount, or absence of the molecular conjugate in a sample. In some embodiments, the presence, amount, or absence of the molecular conjugate in a sample is a measure of the presence, amount, or absence of the molecular conjugate in a single cell or cells. According to some embodiments, methods of the inventive concept include methods of molecular tracking, for example, but not limited to, tracking molecular acquisition/uptake, such as tracking and quantifying antigen uptake, distribution, and/or archiving in a subject and tracking and quantifying antigen uptake, distribution, and/or archiving within cells and/or tissues of the subject. In some embodiments, the cells and/or tissues of the subject in which methods of the inventive concept may be performed include any particular cell or cells of interest in which single-cell nucleic acid sequencing may be performed. In some embodiments, the particular cell or cells of interest may be CD45+ cells. In some embodiments, the particular cell or cells of interest may be CD45− cells. In some embodiments, cells and/or tissues of the subject in which molecular tracking can be performed, for example, tracking antigen distribution and/or archiving, include the lymphatic system, for example, including primary and secondary lymphoid organs and tissues. In some embodiments, molecular tracking, molecular acquisition, antigen uptake, distribution, and/or archiving within cells and/or tissues of the subject are the sites at which lymphocyte activation by antigens takes place, such as the lymph nodes, spleen, and/or Peyer's patches, and cells therein, such as endothelial cells, epithelial cells, and dendritic cells, such as those found in stromal cells, such as lymph node stromal cells, as well as monocytes, T cells and/or B cells. Lymph node stromal cells in which molecular tracking/molecular acquisition/antigen uptake/antigen archiving may be followed/monitored include: fibroblastic reticular cells (FRCs); marginal reticular cells (MRCs); follicular dendritic cells (FDCs); lymphatic endothelial cells (LECs); blood endothelial cells (BECs); alpha-7 integrin pericytes (AlPs); and double negative cells (DNCs). In some embodiments, the particular cell or cells in which molecular tracking/molecular acquisition/antigen uptake/antigen archiving may be followed/monitored include FDCs and/or LECs. Exemplary surface markers expressed by lymph node stromal cells include glycoprotein CD31 and glycoprotein podoplanin GP38. Different sub-populations of lymph node stromal cells are also known by their production of small molecules; where they are located; and their function. Most also express common markers such as desmin, laminin, various subunits of integrins, vascular cell adhesion molecule 1 (VCAM-1) and mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1). LECs in which molecular tracking/molecular acquisition/antigen uptake/antigen archiving may be followed/monitored include, for example, Ptx3 LECs, ceiling LECs, Marco LECs, floor LECs, and/or subcapsular LECs.
Methods of tracking the presence, amount, or absence, and/or tracking change in the presence, amount, or absence of the molecular conjugate in a sample, according to embodiments of the inventive concept, may include methods of single-cell nucleic acid sequencing. The methods of nucleic acid sequencing, such as methods of single-cell nucleic acid sequencing, that may be used in the methods of the inventive concept are not particularly limited, and any suitable nucleic acid sequencing method that would be appreciated by one of skill in the art can be used to sequence the nucleic acids described herein and/or to detect the presence, amount or absence of the various nucleic acids, molecular conjugates, oligonucleotides, amplification products and barcodes described herein. Exemplary methods of single-cell sequencing used in the methods according to the inventive concept include methods of single-cell RNA, in particular mRNA, sequencing. Methods of single-cell mRNA sequencing and methods of analysis of sequencing data/results used in the methods according to the inventive concept include, for example, methods as described in Walsh et al. bioRxiv preprint doi.org/10.1101/2020.08.19.219527v1 (2020), Walsh et al. eLife 10:e62781 doi.org/10.7554/eLife.62781 (2021), and U.S. Patent Application Publication No. 2018/0251825, the disclosures of each of which are incorporated herein by reference.
It will be appreciated that methods of molecular tracking of the inventive concept include tracking of molecules of interest for presence and/or distribution, and changes in presence and/or distribution thereof, within a cell, cells, and/or tissues derived from a subject, such as derived, extracted and/or isolated cells, such as cells derived/extracted/isolated from an organ from the subject, and/or cells derived/extracted/isolated from tissues or tissue samples/tissue slices from the subject, may be performed at multiple/a plurality of time points and/or over a period of time following exposure of the cell, the cells and/or the tissues in a subject and/or derived from a subject to a molecular conjugate including the molecule of interest. The period of time following exposure is not particularly limited, and may be for example, over about 1, 2, 3, 4, 5, 6, 7 days (1 week), 8, 9, 10, 11, 12, 13, or 14 days (2 weeks) after exposure. In some embodiments, the period of time may be about 1, 2, 3, or 4 weeks after exposure. In some embodiments, the period of time may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (1 year) after exposure. In some embodiments, the period of time may be about 1-3 months, about 1-6 months, or about 1-12 months, or even over longer periods of time (years) after exposure.
Subjects suitable in which the composition, compositions and formulations of the present inventive concept may be used, and/or subjects from which samples, such as a cell, cells, tissues, and/or tissue samples for analysis may be derived include, but are not limited to, mammalian subjects. Mammals according to the present inventive concept include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, humans and the like, and mammals in utero. Any mammalian subject in need of using methods according to the present inventive concept is suitable. In some embodiments of the present inventive concept, the subject is a human subject. The human subject treated according to methods of the present inventive concept may be of any gender (for example, male, female or transgender) and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult, elderly).
The nature/manner in which the molecular conjugates, compositions, and/or formulations of the inventive concept may be administered/exposed to a cell, cells, and/or tissues in a subject or derived from a subject is not particularly limited, and any suitable route of administration/exposure of the molecular conjugates, compositions, and/or formulations of the inventive concept (e.g., parenteral, enteral, oral, nasal, inhalational, ocular, transmucosal and/or transdermal, etc.) to a subject, or exposure of the molecular conjugates, compositions, and/or formulations of the inventive concept to a cell, cells and/or tissues in a subject or derived from a subject may be used as would be appreciated by one of skill in the art. Nonetheless, in terms of administration, in some embodiments, the most suitable route may depend on the nature and manner that a subject may be exposed to the antigen/antigenic agent of interest and tracked by the molecular conjugates, compositions, and/or formulations of the inventive concept. In some embodiments, routes of administration are parenteral, including, but not limited to, for example, intravenous, subcutaneous, intramuscular, intradermal, intramucosal, intraperitoneal, etc. administration/injection. In some embodiments, the parenteral administration may by intravenous or intraperitoneal administration. In some embodiments, routes of administration are oral, including, but not limited to, for example, buccal, enteral, sublabial, sublingual, etc. administration. In some embodiments, the routes of administration may be transmucosal administration. In some embodiments, the routes of administration are nasal or intranasal administration. Such administration may be inhalational or direct application to a mucosal membrane.
Having described various aspects of the present inventive concept, the same will be explained in further detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the inventive concept.
Key resources used in these examples are listed in the following table:
Mice. 4-6 week old mice were purchased from Charles River or Jackson Laboratory, unless otherwise stated, bred and housed in the University of Colorado Anschutz Medical Campus Animal Barrier Facility. Wild type and OT1 mice were all bred on a C57BL/6 background. OT1 mice are a TCR transgenic strain specific to the SIINFEKL (SEQ ID NO:1) peptide of ovalbumin (OVA257-264) in the context of H-2Kb. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Colorado.
Phosphorothioate and phosphodiester oligonucleotides. Oligonucleotides were synthesized by Integrated DNA Technologies (IDT) and contained a 5′ amine for conjugation, primer binding site, barcode, 10×Genomics Gel Bead Primer binding site for capture sequence 2, and a 3′ biotin. Phosphorothioated oligonucleotides contained a phosphorothioate modification at every linkage. All oligonucleotide sequences can be found in Supplementary Table 1 from Walsh et al. doi.org/10.1101/2020.08.19.219527v1 (2020), the disclosure of which is incorporated herein by reference. Supplementary Table 1 from Walsh et al. lists antigen tags and other oligonucleotide sequences used in qPCR and single-cell experiments
Conjugation of oligonucleotides to protein. Oligonucleotides were conjugated to ovalbumin by iEDDA-click chemistry89. Oligonucleotides were derivatized with trans-cyclooctene (TCO) in 10× borate buffered saline (BBS; 0.5 M borate, 1.5 M NaCl, pH 7.6; sterile filtered). Dilution of this buffer to 1× results in a final pH of 8.5. A reaction containing 40 nmol of amine-modified oligo (0.5 mM), 1× BBS, 10% DMSO, 8 μL of 100 mM TCO-PEG4-NHS in DMSO (10 mM final; Click Chemistry Tools, A137), pH 8.5 was rotated at room temperature for 15 min. A second aliquot containing the same amount of TCO-PEG4-NHS in DMSO was added and the reaction was rotated at room temperature for another hour. Excess NHS was quenched by adding glycine, pH 8.5 to a final concentration of 20 mM and rotated at room temperature for 5 min. Modification was confirmed by analysis on an 8% denaturing TBE PAGE gel. Samples were precipitated by splitting the reaction into 20 μL aliquots and adding 280 μL of nuclease-free water, 30 μL of 3 M NaCl, and 990 μL of 100% ethanol. The precipitation reaction was incubated at −80° C. overnight followed by centrifugation at >10,000×g for 30 min. The supernatant was discarded, the pellet was washed with 100 μL of 75% ethanol, and centrifuged at >10,000×g for 10 min. The supernatant was removed, and the pellets were dried for 5 min at room temperature. The pellets were recombined by resuspension in 50 μL of 1× BBS. Samples were quantified by A260.
To conjugate methyltetrazine to ovalbumin: detoxified Ovalbumin (Sigma-Aldrich, St. Louis, MO) (using a Triton X-114 lipopolysaccharide detoxification method90), was buffer exchanged into 1× BBS, pH 8.5. To an Amicon 0.5 mL 30 kDa filter (Millipore, UFC5030) was added 1 mg of ovalbumin and 1× BBS to a volume of 450 μL. The filter was centrifuged at 14,000×g for 5 min. The flow through was discarded and the sample washed twice with 400 of 1× BBS. The product-containing column was inverted into a clean collection tube and centrifuged at 1,000×g for 2 min. Assuming no loss, the volume of the sample was adjusted to 2 mg/mL with 1× BBS. 400 μL of 1× BBS was added to the Amicon filter and stored at 4° C. for later use. A 500 μL labeling reaction containing 0.5 mg of ovalbumin in 1× BBS and 50 μL of 2 mM mTz-PEG4-NHS in DMSO (0.2 mM final; Click Chemistry Tools, 1069), pH 8.5 was rotated at 4° C. overnight. Excess NHS was quenched by adding glycine, pH 8.5 to a final concentration of 20 mM and rotated at room temperature for 10 min. The previously stored Amicon filter was centrifuged at 14,000×g for 5 min and the flow through discarded. 400 μL of reaction mixture was added to the filter and centrifuged at 14,000×g for 5 min. This was repeated until all 1 mg of protein had been added to the filter and was supplemented with 1× BBS as needed. Samples were washed 1× with 400 μL of 1× BBS. The product-containing column was inverted into a clean collection tube and centrifuged at 1,000×g for 2 min. Assuming no loss, the volume of the sample was adjusted to 5 mg/mL with 1× BBS.
For the final antigen-DNA conjugation, a 100 μL reaction containing 300 μg of ovalbumin-mTz and 6 nmol of oligonucleotide-TCO (1:1 equivalents) in 1× BBS was rotated at 4° C. overnight. Excess mTz was quenched with 10 μL of 10 mM TCO-PEG4-glycine and rotated at room temperature for 10 min. TCO-PEG4-glycine was prepared by reaction of 10 mM TCO-PEG4-NHS with 20 mM glycine, pH 8.5 in 1× BBS for 1 h at room temperature and stored at −20° C. Products were analyzed by 10% TBE PAGE. For purification, excess ovalbumin and DNA were removed by filter centrifugation. 200 μL of 1× PBS was added to an Amicon 0.5 mL 50 kDa filter (Millipore, UFC5050) followed by 300 μL of sample. The filter was centrifuged at 14,000×g for 5 min and the flow through discarded. Samples were washed five times with 400 of 1× PBS and centrifuged at 14,000×g for 5 min. The product-containing column was inverted into a clean collection tube and centrifuged at 1,000×g for 2 min. Purified products were analyzed by 10% TBE PAGE and total protein quantified with Bio-Rad protein quantification reagent (Bio-Rad, 5000006). LPS contamination after conjugation was below 0.5 EU/mg as mentioned below in the vaccinations section.
Bone marrow derived dendritic cell and lymphatic endothelial cell cultures. Both left and right tibia and femur were isolated under sterile conditions. Bone marrow was extracted from femurs of 6-8-week-old C57BL/6 mice by decollating the top and bottom of the bone and releasing the marrow with 27 gauge syringe and 5 ml of Modified Essential Medium (MEM) (Cellgro). Suspension was strained through 100 μm filter, pressed with the back of a syringe and washed. Cells were spun 1500 RPM, 5 min then suspended in MEM with 10% FBS, 20 ng/ml of GM-CSF from the supernatant of the B78hi-GM-CSF cell line. Every 2 days, dead cellular debris was spun, supernatant collected and combined 1:1 with new 40ng/ml GM-CSF 20% FBS (2×) in MEM. After 7 days of culturing at 37C, 5% CO2, cells were harvested for respective assays. Mouse lymphatic endothelial cells (Cell Biologics, C57-6092) were cultured in Endothelial Cell Media (Cell Biologics, M1168) with kit supplement. T75 Flasks were coated with gelatin for 30 minutes 37° C., washed with PBS and then inoculated with mLEC. Cells were passaged with passive trypsin and split at a density of 1:3. For BMDMs, whole bone marrow was isolated and red blood cells were lysed. Cells were then cultured in M-CSF (50 ng/mL) for 6 days in complete media. Cells were harvested via cell scraper and plated for treatment.
Conjugate detection assay. BMDC and mLEC Cultures were stimulated with 20 μg of anti-CD40, 20 μg Poly I:C, and 5 μg of either OVA-psDNA or OVA in a 6-well format. 24 hr post treatment, cells were washed and refreshed with new media. At designated time points, cells were harvested, counted, and transferred into micro-centrifuge tubes, spun at 350 g, and both supernatant and pellets were frozen at −80° C. Cell pellets were lysed in 50 μL of Mammalian Protein Extraction Reagent (MPER; Thermo Scientific, 78503). Conjugate DNA was measured by qPCR amplification from 1 μL of lysate in a 10 μL reaction containing 5 μL of iTaq Universal SYBR Green Supermix (Bio-Rad, 1725125) and 5 pmol of each primer (Supplementary Table 1 from Walsh et al.). Quantification was measured using an external standard curve and normalized to lysate protein content. To visualize within ova-psDNA acquisition by cells, cells were fixed with 10% formalin for 10 min at room temperature in the dark, washed with PBS, and spun for 10 min at 2000 rpm. Cells were then permeabilized with 100% ice-cold methanol for 20 min at −20° C. Cells were then washed with PBS and spun as above. Cells were stained with the anti-ova antibody as above for at least 2 hr at room temperature and then washed with 1% bovine serum albumin (BSA) with sodium azide (FACS buffer) and spun as above. Cells were then incubated with an anti-rabbit secondary in PE for 1 hr at room temperature and then washed with FACS buffer. Cells were then stained with streptavidin conjugated to BV421 in PBS for 15 min at room temperature and then washed twice with FACS buffer prior to acquiring cells on a FACS CANTO II flow cytometer. Analysis was performed using FlowJo software. Immunofluorescence was performed as above except cells were grown on glass coverslips and stained on cover slips using an anti-rabbit dylight 649 and streptavidin-FITC. Coverslips were mounted with Vectashield with DAPI and imaged on a Zeiss LSM780 confocal microscope. The imaging experiments were performed in the Advanced Light Microscopy Core part of the NeuroTechnology Center at University of Colorado Anschutz Medical Campus
OT1 Isolation and co-culture. CD8 T cells were isolated from an OT1+ mouse using the mojosort mouse CD8 T cell isolation kit (Biolegend) and labeled with violet proliferation dye (BD Biosciences cat #562158). For DC-T cell co-culture, BMDCs were treated with psOVA (5 μg), or OVA+psDNA (5 μg) for 1,3 or 7 days. BMDCs were washed and then co-cultured with labeled OT1s for three days at a 1:10 ratio of BMDC:OT1. Cells were then stained and run on a flow cytometer. OT1 division (percent dividing cells) was calculated as previously described91 using the equation fraction
where i is the generation number (0 is the undivided population), and Ni is the number of events in generation i.
Vaccinations. 6-8 week-old C57BL/6 (CD45.2) mice were immunized with 1E3 or 1E4 colony forming units of Vaccinia Western Reserve or 5 μg of Poly I:C (Invivogen) with or without 5 μg of anti-CD40 (FGK4.5, BioXcell) and 10 μg of OVA-psDNA or OVA in 50 μl volume by footpad injection. Endotoxin levels were quantified using the Pierce Limulus Amebocyte Lysate Chromogenic Endotoxin Quantitiation kit (ThermoScientific) to be less than 0.5 EU/mg for either ovalbumin or ovalbumin conjugated to psDNA.
Nystatin. Nystatin (Sigma N4014) was resuspended in DMSO to a concentration of 10 mg/mL Mice were injected with 50 μL of 10 mg/mL nystatin per footpad one hour prior to injection with ovalbumin conjugated to Alexa 488 (5 μg) in a mixture with polyI:C and anti-CD40 (2.5 μg each). LNs were harvested and digested as below (preparation of single cell suspensions) and stained with CD45 brilliant violet 510 (Biolegend clone 30F11, 1:300), PDPN APC (Biolegend clone 8.1.1, 1:200), CD31 PercP Cy5.5 (Biolegend clone 390, 1:200) and PD-L1 pacific blue (Biolegend clone 10F.9G2, 1:200).
Tetramer and intracellular cytokine assays. Draining LNs were processed by glass slide maceration 7 days after injection, washed and suspended in FACS (2% FBS in PBS) buffer containing Tetramer (OVA257-264)-PE (1:400) (NIH tetramer core facilty), CD8 APC-Cy7 (Biolegend clone 53-6.7 1:400) for 1 hr at 37° C. Cells were washed and stained for 30 minutes in CD44 PerCP Cy5.5 (Biolegend clone IM7, 1:400), B220 BV510 (Biolegend clone RA3-6B2, 1:300). Samples were run on the FACs Canto II flow cytometer (BD).
Preparation of single-cell suspensions. Two days or two weeks following vaccination with 1E3 CFU of VV-WR with 10 μg is of ova-psDNA per foot pad, popliteal LNs were removed from 15 mice and LNs were pulled-apart with 22-gauge needles. Tissue was digested with 0.25 mg of Liberase DL (Roche, Indianapolis, IN) per ml of EHAA media with DNAse (Worthington, Lakewood, NJ) at 37 degrees. Every 15 minutes media was removed, cells spun down and new digestion media added to the undigested tissue until no tissue remained, ˜1 hr. Following digestion cells were filtered through a screen and washed with 5 mM EDTA in EHAA. LN cells were then divided into thirds where one third underwent staining with CD11c (N418), CD11b and B220 and a live/dead dye (Tonbo). Live cells were then sorted into 4 tubes on a FACs Aria Cell Sorter (BD): sorted CD11c-APC Cy7 (Biolengend clone N418 1:400)+ cells, sorted CD1lb PE-Cy7 (Biolegend clone M1/70)+ cells, sorted B220 PE (Biolegend clone RA3-6B2)+ cells and Fixable Viability Stain 510 (BD Biosciences Cat #546406) ungated live cells which were recombined at a 4:4:1:1 ratio, respectively. For the remaining two-thirds of cells, cells were stained with CD45 PE followed by magnetic bead isolation using the Miltenyi bead isolation kit. CD45 negative cells that passed through the column were then washed. Both sorted and selected (CD45+ and CD45=) cells were then washed with PBS in 0.1% BSA as described in the Cell Prep Guide (10× Genomics) and counted using a hemacytometer. Final concentration of cells was approximately 1000 cells/ul and approximately 10-20 μL were assayed.
Single-cell library preparation using the 10× Genomics platform. Cells were assayed using the 10× Genomics single-cell 3′ expression kit v3 according to the manufacturer's instructions (CG000183 Rev B) and CITE-seq protocol (cite-seq.com/protocol Cite-seq_190213) with the following changes:
Transcriptome and oligonucleotide detection and analysis. Briefly, FASTQ files from the gene expression and antigen tracking libraries were processed using the feature barcode version of the cellranger count pipeline (v3.1.0). Reads were aligned to the mm10 and Vaccinia virus (NC_006998) reference genomes. Analysis of gene expression and antigen tracking data was performed using the Seurat R package (v3.2). Antigen tracking and gene expression data were combined into the same Seurat object for each sample (CD45−/day 2, CD45+/day 2, CD45−/day 14, CD45+/day 14). Cells were filtered based on the number of detected genes (>250 and <5000) and the percent of mitochondrial reads (<15%). Gene expression counts were log normalized (NormalizeData), and relative ova signal was calculated by dividing ova-psDNA counts by the median ova-psDNA counts for all T and B cells present in the sample. To allow for the values to be log-transformed for visualization, a pseudo count was added (smallest non-zero value*0.5).
Gene expression data were scaled and centered (ScaleData). 2000 variable features (Find VariableFeatures) were used for PCA (RunPCA) and the first 40 principal components were used to find clusters (FindNeighbors, FindClusters) and calculate uniform manifold approximation and projection (UMAP) (RunUMAP). Cell types were annotated using the R package clustifyr (rnabioco.github.io/clustifyr)48 along with reference bulk RNA-seq data from ImmGen (available for download through the clustifyrdata R package, rnabioco.githublo/clustifyrdata). To annotate cell subtypes, the samples were then divided into separate objects for DCs, LECs, and FRCs. Cell subsets were then annotated using clustifyr with reference bulk RNA-seq data for DCs49,50, FRCs51, and LECs52,53,54. After assigning DC, LEC, and FRC subtypes, the other cell types (TB cells, epithelial cells, NK cells) were added back to the objects and reprocessed as described above.
Identification of ova-low and -high populations was accomplished using a two-component Gaussian mixture model implemented with the R package mixtools (cran.r-project.org/web/packages/mixtools/index.html). All LECs were used when identifying ova-low and ova-high cells (
Raw data and analysis software. Raw and processed data for this study have been deposited at NCBI GEO under accession GSE150719. A reproducible analysis pipeline is available at github.com/rnabioco/antigen-tracking.
Statistical analysis. Statistical analysis was done using either a non-parametric 2 tailed Mann-Whitney t-test or multiple t-tests with a two stage step up method of Benjamin, Krieger and Yekutieli without assuming consistent standard deviations. A biologic replicate was considered a measurement of a biologically distinct sample (such as a separate mouse), a technical replicate was considered a repeated measurement of the same sample. Each in vivo analysis was performed with 3-6 mice per group as determined by a power calculation using the assumption (based on prior data) that there will be at least a two-fold change with a standard deviation of less than 0.5. To calculate numbers, a power calculation was performed with an alpha of 0.5 and a 1-beta of 0.80 to determine at least 3 mice per group should be evaluated. Error bars indicate the standard error of the mean (SEM) and all analysis was blinded.
To quantify the dissemination and uptake of antigen in the draining lymph node (LN) after vaccination, a strategy was developed to measure antigen levels using single-cell mRNA sequencing. Many prior studies have used the model antigen, ovalbumin (ova), conjugated to a fluorophore to track antigen in vivo. Here, ova to DNA oligonucleotides with barcodes were conjugated that were suitable for analysis by single-cell mRNA sequencing (
To determine whether conjugation of psDNA to ovalbumin affected ovalbumin processing and presentation, BMDC presentation of ova-derived SIINFEKL (SEQ ID NO:1, OVA257-264) peptide was measured by co-culture with OVA257-264-specific OT1 T cells. BMDCs given ova-psDNA induced significantly more proliferation of OT1 T cells than unconjugated ovalbumin (
It was next asked whether vaccination with ova-psDNA conjugates elicits a T cell response in vivo. Antigen-specific T cell responses were compared in mice vaccinated with a mixture of ova-psDNA and polyI:C/αCD40 to its individual components (ova, psDNA, polyI:C and polyI:C/αCD40;
It has been previously shown that a vaccination strategy comprised of soluble antigen and vaccinia virus (Western Reserve; VV) induced robust antigen archiving that lasts longer than those using polyI:C/αCD40 adjuvant12. To evaluate antigen-psDNA performance during an active infection, T cell responses after vaccination were determined by comparing individual components with mixtures of ova, VV, ova-pDNA, or ova-psDNA. Subcutaneously administered ova-psDNA alone again elicited a T cell response (
Given the ability of the antigen-psDNA conjugates to induce a robust immune response in vivo (
A total of 800 cells were recovered in the CD45− fraction and 8,187 cells in the CD45+ fraction at the 2 day time point. More CD45− cells (6,372 CD45−; 4,840 CD45+) were recovered at the 14 day time point likely due to expansion and proliferation of the lymph node stoma14,22,47. Cell types were classified using an automated approach48, comparing measured mRNA expression patterns to reference data sets for DCs49,50, FRCs51, and LECs52,53,54 (Supplementary Table 2 from Walsh et al. doi.org/10.1101/2020.08.19.219527v1 (2020), the disclosure of which is incorporated herein by reference). Supplementary Table 2 from Walsh et al. lists the results from comparing relative ova signal for cell types shown in
The dynamic changes of myeloid populations were firs examined in the lymph node. Conventional DCs were detected, including cDC1 and cDC2 (
As expected, at day 2 a large population was identified of LN resident cDC2B (cDC2 Tbet−) cells harboring ova-psDNA49. However, cDC2A (cDC2 Tbet+) cells were not found, consistent with their role in anti-inflammatory processes49. The myeloid populations contained CCR7hi cDCs (n=3,432; 42% of total), which were classified as migratory DCs. This migratory DC population included Langerhans cells (n=285; 3.5% of total), migratory cDC1s (n=593; 7.2% of total), and migratory cDC2s (n=2,554; 31% of total) 50 , migrating from the dermis (
Using unique barcodes, the amount of ova-psDNA, psDNA, and pDNA across cell types was quantified. Levels of ova-psDNA molecules spanned four orders of magnitude, ranging up to 104 unique molecules and depending on the cell types and time point (
At the early day 2 time point, LN resident cDC2s contained high levels of antigen-psDNA, consistent with studies of soluble antigens2 (
Next, antigen levels in the LN stromal cell populations were examined (
At the day 14 time point, several LEC subtypes maintained high antigen levels (
Similar to the endothelial cell population, the number and types of non-endothelial stromal cells increased at the later time point after immunization. Non-endothelial stromal cells in the lymph node are classified by their location in the lymph node into T-zone reticular cells (TRC), marginal reticular cells (MRC), follicular dendritic cells (FDC), and perivascular cells (PvC)51. Recently, additional subsets were identified including: Ccll9lo TRCs located at the T-zone perimeter, Cxcl9+ TRCs found in both the T-zone and interfollicular region, CD34+ stromal cells found in the capsule and medullary vessel adventitia, indolethylamine N-methyltransferase+ stromal cells found in the medullary chords, and Nr4a1+ stromal cells51.
At the early time point, the Cxcl9+ TRCs and CD34+ SCs51, had high amounts of antigen (˜10-fold relative to TB cells) (
Finally, these data provided insight into antigen transfer between stromal and dendritic cells, a process important for enhanced protective immunity12,14. It was previously shown that archived antigen is transferred from LECs to migratory Batf3-dependent cDC1s two weeks after infection12. Here, it was confirmed that CCR7hi migratory cDC1s had the highest amount of antigen two weeks after vaccinia infection (
Gene Expression Signatures Associated with Antigen Acquisition by Dendritic Cells.
Next, the variation in antigen levels across cell types was leveraged (
At the early time point, genes upregulated in antigen-high DCs confirmed DC activation (Supplementary Table 4 from Walsh et al.). Antigen-high cDC2 Tbet− cells upregulated genes Ccl2 and Cxcl2 (consistent with active recruitment of inflammatory cells66,69), Msr1 (consistent with antigen scavenging71), as well as Pkm, Lgals3, and Mif (consistent with DC-T cell responses and DC differentiation during inflammation36,65,68) (
At the late day 14 time point, the highest antigen counts were found in the migratory cDC1 population, consistent with a role for migratory cDC1s in archived antigen acquisition from LECs12 (
Gene Expression Signatures Associated with Antigen Archival by LECs.
Next, the LEC population was evaluated to determine whether the classification approach could identify genes involved in antigen archiving. The classifier was applied to LECs as a population and found large numbers of antigen-high-floor, collecting, and ceiling LECs (
Using this classification approach, 142 mRNAs were identified that were significantly changed in antigen-high or antigen-low LECs (Supplementary Table 3 from Walsh et al.). Supplementary Table 3 from Walsh et al. lists genes associated with ova-high cells for DCs, FRCs, and LECs. Ova-low and ova-high cells were independently identified for DCs, FRCs, and LECs using a gaussian mixture model implemented with the R package mixtools. Differentially expressed genes were identified using a Wilcoxon rank sum test performed using the R package presto (wilcoxauc). The Benjamini-Hochberg method was used to correct for multiple comparisons. Genes were filtered to only include those with an adjusted p-value<0.05, log fold change>0.25, auc>0.5, and with at least 50% of ova-high cells expressing the gene. The average expression, log fold change, test statistic (statistic), area under the receiver operator curve (auc), percentage of ova-high cells that express the gene (pct_in), and percentage of ova-low cells that express the gene (pct out) are included. Prox1, while expressed by all LECs identified, was highly expressed in antigen-high LECs, independent of the LEC type (
Upregulation of Cavin1 and Cavin2 by antigen-high LECs suggested that caveolar endocytosis may contribute to antigen acquisition by LECs, consistent with LEC dynamin-mediated transcytosis in vitro78 (
Finally, expression of Stabilin-1 (Stab1) and Stabilin-2 (Stab2) is increased in antigen-high LN endothelial cells, suggesting that scavenging pathways are required for the acquisition of antigen-psDNA conjugates after vaccination. Stab2 is uniquely expressed by LECs in the lymph node and not by BECs79, and Stabilin-1 and Stabilin-2 act as receptors for internalization of antisense oligonucleotides with phosphorothioate linkages in liver endothelial cells and Kupffer cells80. However, significant amounts of unconjugated psDNA were not found in LECs (
Development of a “molecular tracking device” enables tracking of antigen throughout the lymph node to specific cell types that acquire and archive antigens following subcutaneous immunization. Previous work used canonical surface markers to track antigen by microscopy and flow cytometry; instead, this approach simultaneously defines cell type by gene expression and quantifies the acquired antigen. The molecular tracking device enables the study of archived antigens at time points beyond the lifetime of antigen-fluorophore conjugates and provides a more complete catalog of cell types involved in antigen acquisition and retention.
This approach validates and expands upon previous work on antigen archiving and cell types that enhance protective immunity. Both here and in previous work, it was found that whereas LECs archive antigen, migratory DCs passing through the lymphatic vasculature are required to retrieve and present archived antigen to memory CD8 T cells derived from the initial infection or immunization s . Antigen exchange from LECs to DCs and subsequent DC presentation yields memory CD8 T cells with robust effector function during infectious challenge. Several recent reports defined LEC and non-endothelial stromal cell subsets within the lymph node51,52,53,54. By combining the molecular tracking device described in this work with these reference cell types, it was found that non-endothelial stromal cell types acquire foreign antigens including CD34+ stromal cells, which neighbor subcapsular sinus LECs in the tissue51. These findings indicate that the interstitial pressure created by subcutaneous vaccination allows antigens to pass through the tissue directly to the LN capsule, bypassing the lymphatic capillaries. Intriguingly, bypass of lymphatic capillaries may still lead to LEC acquisition of antigens from the CD34+ stromal cells via SC-LEC exchange. Such a mechanism would encourage future LEC-DC interactions and provide a benefit to protective immunity.
Molecular tracking devices provide a measure of cell state orthogonal to gene expression, which was leveraged to identify candidate pathways involved in antigen acquisition (
The psDNA component of the tracking device elicits an immune response similar to other TLR-antigen conjugate vaccines82,83, likely due antigen-psDNA stability within dendritic cells that causes prolonged antigen presentation in the cells that acquire the antigen45,41. This effect is illustrated by increased IFNγ production in the absence of ex vivo peptide stimulus (ova-psDNA compared to unconjugated ova;
Molecular tracking devices enable new approaches to study molecular dissemination in vivo. To date, protein-DNA conjugates have been deployed in single-cell mRNA sequencing experiments for ex vivo staining applications (e.g., CITE-seq46). This work lays the groundwork for molecular tracking devices involving protein, antibody, drug, or pathogens conjugated to nuclease-resistant, barcoded oligonucleotides that are stable during transit through animal tissues. The approach naturally extends to understanding how multiple different antigens are processed (using unique DNA barcodes) and enables studies to manipulate antigen archiving to improve vaccines, vaccine formulations, and prime-boost strategies. Moreover, the oligonucleotide portion of the tracking device can enable analysis of its distribution in cells by in situ hybridization or intact tissue by spatial transcriptomics86,87,88, obviating the need for antibody-mediated detection of antigen.
Whereas specific embodiments of the present inventive concept have been shown and described, it will be understood that other modifications, substitutions, and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the inventive concept, which should be determined from the appended claims.
This application claims priority to U.S. Provisional Application Ser. No. 63/136,798, filed Jan. 13, 2021, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with Government Support under Federal Grant Nos. RO1 AI121209, T32 A1007405, R35 GM119550, T32 A1074491, and R21 AI155929, awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/12235 | 1/13/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63136798 | Jan 2021 | US |
