Patent Description:
The adaptive immune system is directed through specific interactions between immune cells and antigen-presenting cells (e.g. dendritic cells, B-cells, monocytes and macrophages) or target cells (e.g. virus infected cells, bacteria infected cells or cancer cells). In important field in immunology relates to the understanding of the molecular interaction between an immune cell and the target cell.

Specifically for T-lymphocytes (T-cells), this interaction is mediated through binding between the T-cell receptor (TCR) and the Major Histocompatibility Complex (MHC) class I or class II. The MHC molecules carries a peptide cargo, and this peptide in decisive for T-cell recognition. The understanding of T-cell recognition experienced a dramatic technological breakthrough when Atman et al. (<NUM>) in <NUM> discovered that multimerization of single peptide-MHC molecules into tetramers would allow sufficient binding-strength (avidity) between the peptide-MHC molecules and the TCR to determine this interaction through a fluorescence label attached to the MHC-multimer. Such fluorescent-labelled MHC multimers (of both class I and class II molecules) are now widely used for determining the T-cell specificity. The MHC multimer associated fluorescence can be determined by e.g. flow cytometry or microscopy, or T-cells can be selected based on this fluorescence label through e.g. flow cytometry or bead-based sorting. However, a limitation to this approach relates to the number of different fluorescence labels available, as each fluorescence label serve as a specific signature for the peptide-MHC in question.

Thus, this strategy is poorly matching the enormous diversity in T-cell recognition. For the most predominant subset of T-cells (the αβ TCR T-cells), the number of possible distinct αβ TCRs has been estimated at ~<NUM><NUM> (<NUM>) although the number of distinct TCRs in an individual human is probably closer to <NUM><NUM> (<NUM>). Therefore, much effort has attempted to expand the complexity of the T-cell determination, with the aim to enable detection of multiple different T-cell specificities in a single sample. A more recent invention relates to multiplex detection of antigen specific T-cells is the use of combinatorial encoded MHC multimers. This technique uses a combinatorial fluorescence labelling approach that allows for the detection of <NUM> different T-cell populations in a single sample when first published (<NUM>,<NUM>), but has later been extended through combination with novel instrumentation and heavy metal labels to allow detection of around <NUM> different T-cell populations in a single sample (<NUM>).

The requirement for new of technologies that allow a more comprehensive analysis of antigen-specific T-cell responses is underscored by the fact that several groups have tried to develop so-called MHC microarrays. In these systems, T-cell specificity is not encoded by fluorochromes, but is spatially encoded (<NUM>,<NUM>). In spite of their promise, MHC microarrays have not become widely adopted, and no documented examples for its value in the multiplexed measurement of T-cell responses, for instance epitope identification, are available.

<CIT> discloses multiplex sorting of cells and subsequent analysis of said sorted cells, including detection of T cells.

<CIT> discloses methods of preparing DNA-encoded chemical libraries.

<CIT> and <CIT> disclose methods to identify single compounds using a DNA barcode.

<CIT> ("<NPL>) disclose the use of PCR amplification to identify molecules tagged by a barcode flanked by conserved primers.

Kwong, Gabriel Agner ('<NUM>') ("DNA Encoded Biotechnologies for Informative cancer Diagnostics", Dissertation (Ph. ), California Institute of Technology) discloses detection of T cells using cellular barcoding including a step of cleaving barcodes from the capture agent before their amplification.

Considering the above, there remains a need for a high-throughput method in the art of detection, isolation and/or identification of specific antigen responsive cells, such as antigen specific T-cells.

Further, there remains a need in the art, considering the often limited amounts of sample available, for methods allowing detection, isolation and/or identification of multiple species of specific antigen responsive T-cells in a single sample.

The present disclosure relates to the use of nucleic acid-barcodes for the determination and tracking of antigen specificity of immune cells.

In an aspect of the present disclosure a nucleic acid-barcode will serve as a specific label for a given peptide-MHC molecule that is multimerized to form a MHC multimer. The multimer can be composed of MHC class I, class II, CD1 or other MHC-like molecules. Thus, when the term MHC multimers is used below this includes all MHC-like molecules. The MHC multimer is formed through multimerization of peptide-MHC molecules via different backbones. The barcode will be co-attached to the multimer and serve as a specific label for a particular peptide-MHC complex. In this way up to <NUM> to <NUM> (or potentially even more) different peptide-MHC multimers can be mixed, allow specific interaction with T-cells from blood or other biological specimens, wash-out unbound MHC-multimers and determine the sequence of the cell-bound DNA-barcodes. When selecting a cell population of interest, the sequence of barcodes present above background level, will provide a fingerprint for identification of the antigen responsive cells present in the given cell-population. The number of sequence-reads for each specific barcode will correlate with the frequency of specific T-cells, and the frequency can be estimated by comparing the frequency of reads to the input-frequency of T-cells. This strategy may expand our understanding of T-cell recognition.

The DNA-barcode serves as a specific labels for the antigen specific T-cells and can be used to determine the specificity of a T-cell after e.g. single-cell sorting, functional analyses or phenotypical assessments. In this way antigen specificity can be linked to both the T-cell receptor sequence (that can be revealed by single-cell sequencing methods) and functional and phenotypical characteristics of the antigen specific cells.

Furthermore, this strategy may allow for attachment of several different (sequence related) peptide-MHC multimers to a given T-cell - with the binding avidity of the given peptide-MHC multimer determining the relative contribution of each peptide-MHC multimer to the binding of cell-surface TCRs. By applying this feature it is possible to allow the determination of the fine-specificity/consensus recognition sequence of a given TCR by use of overlapping peptide libraries or alanine substitution peptide libraries. Such determination is not possible with current MHC multimer-based technologies.

Thus, one aspect of the disclosure relates to a multimeric major histocompatibility complex (MHC) comprising.

Another aspect of the present disclosure relates to a composition comprising a subset of multimeric major histocompatibility complexes (MHC's) according to the disclosure, wherein each set of MHC's has a different peptide decisive for T cell recognition and a unique "barcode" region in the DNA molecule.

Yet another aspect of the present disclosure is to provide a kit of parts comprising.

Still another aspect of the present disclosure is to provide a method for detecting antigen responsive T cells in a sample comprising:.

wherein said binding is detected by amplifying by PCR the barcode region of said nucleic acid molecule linked to the one or more cell-bound multimeric MHC's; and sequencing of said amplified barcode regions.

Further aspects relates to different uses.

The present disclosurewill now be described in more detail in the following.

Any embodiment not falling under the scope of the appended claims does not form part of the invention.

Prior to discussing the present disclosure in further details, the following terms and conventions will first be defined:.

In the present context, a nucleic acid barcode is a unique oligo-nucleotide sequence ranging for <NUM> to more than <NUM> nucleotides. The barcode has shared amplification sequences in the <NUM>' and <NUM>' ends, and a unique sequence in the middle. This sequence can be revealed by sequencing and can serve as a specific barcode for a given molecule.

In the present aspect it is understood that sequencing also relates to e.g. deep-sequencing or next-generation sequencing, in which the amplified barcodes (the PCR product) is sequenced a large number of repetitive time (number of total reads, e.g. <NUM> of reads). The number of reads for the individual barcode sequence will relate to their quantitative presence in the amplification product, which again represents their quantitative presence before amplification, since all DNA-barcodes have similar amplification properties. Thus, the number of reads for a specific barcode sequences compared to the total number of reads will correlate to the presence of antigen responsive cells in the test-sample.

Referring now to the disclosure in more detail, <FIG> describes how peptide-MHC molecules, nucleic acid (DNA)-barcodes and (optional) fluorescent labels are assembled to form a library of MHC multimers each holding a DNA-barcode specific for the given peptide-MHC molecule involved. <FIG>) the barcode is designed to have a unique sequences that can be determined through DNA sequencing. Also the barcode have shared amplification ends, enabling amplification of all DNA-barcodes simultaneously in a PCR reaction. DNA-barcodes are attached to the MHC-multimerization backbone (e.g. via a biotin linker binding to streptavidin on the multimer backbone). <FIG> represents the multimer backbone. This may be any backbone that allow multimerization of macromolecules. The backbone may (optionally) hold a fluorescence label (illustrated by the asterisk) to track the total pool of MHC multimer binding cells irrespectively of the peptide-MHC multimer specificity. <FIG> represents the peptide-MHC molecule of interest, carrying a specific peptide cargo (horizontal line). <FIG> represents the assembled peptide-MHC multimers carrying the DNA barcode.

An aspect of the disclosure relates to a multimeric major histocompatibility complex (MHC) comprising.

Different types of backbones may be used. Thus, in an embodiment of the disclosure the backbone molecule is selected from the group consisting of polysaccharides, such as glucans such as dextran, a streptavidin or a streptavidin multimer. The skilled artisan may find other alternative backbones.

The MHC's may be coupled to the backbone by different means. Thus, in an embodiment of the disclosure the MHC's are coupled to the backbone through a streptavidin-biotin binding. Again other binding moieties may be used. The specific binding may use specific couplings points. In another embodiment of the disclosure the MHC's are linked to the backbone via the MHC heavy chain.

The MHC consists of different elements, which may partly be expressed and purified from cell systems (such as the MHC heavy chain and the Beta-<NUM>-microglobulin element). Alternatively, the elements may be chemically synthesized. The specific peptide is preferably chemically synthesized.

All three elements are required for the generation of a stable MHC (complex). Thus, in an embodiment of the disclosure the MHC is artificially assembled.

The multimeric MHC may comprise different numbers of MHC's. Thus, in yet an embodiment of the disclosure the multimeric major histocompatibility complex (MHC) is composed of at least four MHC's, such as at least eight, such as at least ten, <NUM>-<NUM>, <NUM>-<NUM>, such as <NUM>-<NUM> or such as <NUM>-<NUM> MHC's.

The nucleic acid component (preferably DNA) has a special structure. Thus, in an embodiment of the disclosure the at least one nucleic acid molecule is composed of at least a <NUM>' first primer region, a central region (barcode region), and a <NUM>' second primer region. In this way the central region (the barcode region) can be amplified by a primer set. The length of the nucleic acid molecule may also vary. Thus, in another embodiment of the disclosure the at least one nucleic acid molecule has a length in the range <NUM>-<NUM> nucleotides, such as <NUM>-<NUM>, such as <NUM>-<NUM>, such as <NUM>-<NUM> nucleotides. The coupling of the nucleic acid molecule to the backbone may also vary. Thus, in a further embodiment of the disclosure the at least one nucleic acid molecule is linked to said backbone via a streptavidin-biotin binding. Other coupling moieties may also be used.

In a further embodiment of the disclosure the at least one nucleic acid molecule comprises or consists of DNA, RNA, and/or artificial nucleotides such as PLA or LNA. Preferably DNA, but other nucleotides may be included to e.g. increase stability.

Different types of MHC's may form part of the multimer. Thus, in an embodiment of the disclosure the MHC is selected from the group consisting of class I MHC, a class II MHC, a CD1, or a MHC-like molecule. For MHC class I the presenting peptide is a <NUM>-11mer peptide; for MHC class II, the presenting peptide is <NUM>-18mer peptides. For alternative MHC-molecules it may be fragments from lipids or gluco-molecules which are presented.

It may also be advantageously if it was possible to determine the complete pool of bound multimers when incubated with a sample (of cells). Thus, in a preferred embodiment of the disclosure, the backbone further comprises one or more linked fluorescent labels. By having such coupling better quantification can be made. Similar the labelling may be used for cell sorting.

<FIG> illustrates the generation of a full barcode library. <FIG>, this library is composed of multiple, potentially more than <NUM> different peptide-MHC multimers, each with a specific DNA-barcode. Such that barcode#<NUM> codes for peptide-MHC complex#<NUM>, barcode#<NUM> codes for peptide-MHC complex#<NUM>, barcode#<NUM> codes for peptide-MHC complex#<NUM>, and so on until the possible mixture of thousands different specificities each with a specific barcode. <FIG> represents the final reagent, which is a mixture of numerous different MHC-multimers each carrying a specific DNA barcode as a label for each peptide-MHC specificity.

As previously described a pool (library) of different sets of multimeric major histocompatibility complexes (MHC's) may be used to analyze an overall cell population for its specificity for peptides. Thus, another aspect of the disclosure relates to a composition comprising a subset of multimeric major histocompatibility complexes (MHC's) according to the disclosure, wherein each set of MHC's has a different peptide, decisive for T cell recognition and a unique "barcode" region in the DNA molecule. In the present context, it is to be understood that each specific multimeric major histocompatibility complex is present in the composition with a certain number and that there is subset of different multimeric major histocompatibility complexes present in the composition.

Preferably all specific region for each multimeric MHC can be determined with only a few primer sets, preferably only one primer set. Thus, in an embodiment of the disclosure the primer regions in the DNA molecule are identical for each set of MHC's. In this way only one primer set is required. In an alternative embodiment of the disclosure, the multimeric MHC's are grouped by different primer sets, thereby allowing multiplication of different sets of the cell-bound multimeric MHCs. In this way background noise may be limited, while also retrieving information of specific bindings. Thus, different primer set for different sets of cell-bound MHC's may be used.

The number of individual sets of multimeric MHC's may vary. Thus, in an embodiment of the disclosure the composition comprises at least <NUM> different sets of multimeric MHC's such as at least <NUM>, such as at least <NUM>, at least <NUM>, at least <NUM>, such as in the range <NUM>-<NUM>, such as <NUM>-<NUM> or such as <NUM>-<NUM> sets of MHC's.

The composition of the disclosure may form part of a kit. Thus, yet an aspect of the disclosure relates to a kit of parts comprising.

In <FIG> it is illustrated how this library can be used for staining of antigen responsive cells in a single sample. <FIG>, cells in single cell suspension (may e.g., but not exclusive, originate from peripheral blood, tissue biopsies or other body fluids) are mixed with the peptide-library represented in <FIG>. <FIG>, after staining, cells are sequentially washed and spun to remove residual MHC multimers that are not bound to a cellular surface. Specific cell populations, e.g. T-cells (CD8 or CD4 restricted), other immune cells or specifically MHC multimer binding T-cells may be sorted by flow cytometry or others means of cell sorting/selection. <FIG>, the DNA-barcode oligonucleotide sequences bound to the cell population is amplified by PCR. Fig 2D, this amplification product is sequenced by deep sequencing (providing <NUM>-<NUM> of reads). The sequencing will reveal the specific barcode sequence of DNA barcodes attached to cells in the specimen after selection, as these will appear more frequent than sequences associated to the background of non-specific attachment of MHC multimers. The "signal-to-noise" is counteracted by the fact that any unspecific MHC multimer event will have a random association of <NUM>/<NUM> different barcodes (dependent of the size of the library), making it even more sensitive than normal multimer staining.

Through analyses of barcode-sequence data, the antigen specificity of cells in the specimen can be determined. When DNA-barcode#<NUM> is detected above background level of reads it means that peptide-MHC multimer#<NUM> was preferentially bound to the selected cell type. Same goes for barcode no. <NUM>, <NUM>, <NUM>, <NUM>,. etc. up to the potential combination of more than <NUM> (nut not restricted to this particular number). When the number of input cells are known, e.g. when cell populations of interest is captured via a fluorescence signal also attached to the multimer by flow cytometry-based sorting or other means of capturing/sorting, the specific T-cell frequency can be calculated comparing the frequency of barcode-reads to the number of sorted T-cells.

Therefore, the multimeric MHC's and/or the compositions according to the disclosure may be used for different purposes. Thus, yet another aspect of the disclosure relates to a method for detecting antigen responsive T cells in a sample comprising:.

wherein said binding is detected by amplifying by PCR the barcode region of said nucleic acid molecule linked to the one or more cell-bound multimeric MHC's (through the backbone); and sequencing of said amplified barcode regions.

In an embodiment of the disclosure the method includes providing the (biological) sample.

As known to the skilled person, unbound molecules should be removed. Thus, in an embodiment of the disclosure unbound (multimeric) MHC's are removed before amplification, e.g. by washing and/or spinning e.g. followed by removing of the supernatant.

The type of sample may also vary. In an embodiment of the disclosure the sample is a biological sample. In an embodiment of the disclosure the sample is a blood sample, such as an peripheral blood sample, a blood derived sample, a tissue biopsy or another body fluid, such as spinal fluid, or saliva. The source of the sample may also vary. Thus, in a further embodiment of the disclosure said sample has been obtained from a mammal, such as a human, mouse, pigs, and/or horses.

It may also be advantageously to be able to sort the cells. Thus, in an embodiment of the disclosure the method further comprises cell sorting by e.g. flow cytometry such as FACS. This may e.g. be done if the backbone is equipped with a fluorescent marker. Thus, unbound cells may also be removed/sorted.

As also known to the skilled person, the measured values are preferably compared to a reference level. Thus, in an embodiment of the disclosure said binding detection includes comparing measured values to a reference level, e.g. a negative control and/or total level of response in the sample. In a further embodiment of the disclosure, said amplification is PCR such as QPCR.

As also previously mentioned the detection of the barcode includes sequencing of the amplified barcode regions. Thus, in an embodiment of the disclosure the detection of barcode regions includes sequencing of said barcode region, such as by deep sequencing or next generation sequencing.

In <FIG>, it is illustrated how this technology can be used to link different properties to the antigen specificity of a cell population. illustrates how cells after binding to a barcode labeled MHC multimer library may be exposed to a certain stimuli. Cell populations can be selected based on the functional response to this stimuli (e.g., but not exclusive, cytokine secretion, phosphorylation, calcium release or numerous other measures). After selecting the responsive or non-responsive population (following the steps of <FIG>), the DNA barcodes linked to the cell-bound multimeric MHCs can be sequenced to decode the antigen responsiveness, and thereby determining the antigen-specificities involved in a given response.

<FIG> illustrates how cells can be selected based on phenotype, to link a certain set of phenotypic characteristics to the antigen-responsiveness.

<FIG> represents the possibility for single-cell sorting of MHC-multimer binding cells based on the co-attached fluorescence label on the MHC multimer. Through single-cell sorting the antigen-specificity of the given cell can be determined on a single cell level through sequencing of the associated barcode label. This can be linked to the TCR that can also be sequenced on a single cell level, as recently described (<NUM>). Hereby, this disclosure will provide a link between the TCR sequence, or other single-cell properties and the antigen specificity, and may through the use of barcode labeled MHC multimer libraries enable definition of antigen-specific TCRs in a mixture of thousands different specificities.

<FIG> illustrates the use of barcode labeled MHC multimer libraries for the quantitative assessment of MHC multimer binding to a given T-cell clone or TCR transduced/transfected cells. Since sequencing of the barcode label allow several different labels to be determined simultaneously on the same cell population, this strategy can be used to determine the avidity of a given TCR relative to a library of related peptide-MHC multimers. The relative contribution of the different DNA-barcode sequences in the final readout is determined based on the quantitative contribution of the TCR binding for each of the different peptide-MHC multimers in the library. Via titration based analyses it is possible to determine the quantitative binding properties of a TCR in relation to a large library of peptide-MHC multimers. All merged into a single sample. For this particular purpose the MHC multimer library may specifically hold related peptide sequences or alanine-substitution peptide libraries.

<FIG> shows experimental data for the feasibility of attaching a DNA-barcode to a MHC multimer and amplify the specific sequences following T-cell staining. <FIG> shows the staining of cytomegalovirus (CMV) specific T-cells in a peripheral blood samples. The specific CMV-derived peptide-MHC multimers was labeled with a barcode (barcode#<NUM>) and mixed with an irrelevant/non-specific peptide-MHC multimer labeled with barcode (barcode#<NUM>) and mixed with <NUM> other non-barcode labeled non-specific MHC multimers. Data here shows the feasibility for staining of CMV-specific T-cells in a mixture of <NUM> other MHC multimers. Data is shown for three different staining protocols. <FIG> shows the readout of the specific barcode sequences by quantative PCR. Barcode#<NUM> (B#<NUM>) determining the CMV specific T-cell in detected for all three staining protocols, whereas the irrelevant/non-specific barcode signal, barcode#<NUM> (B#<NUM>) is undetectable.

Overall, the multimeric MHC's or compositions comprising such sets of MHC's may find different uses. Thus, an aspect relates to the use of a multimeric major histocompatibility complex (MHC) or a composition according to the disclosure for the detecting of antigen responsive T cells in a sample.

Another aspect relates to the use of a multimeric major histocompatibility complex (MHC) or a composition according to the disclosure in the diagnosis of diseases or conditions, preferably cancer and/or infectious diseases.

A further aspect relates to the use of a multimeric major histocompatibility complex (MHC) or a composition according to the disclosure in the development of immune-therapeutics.

Yet a further aspect relates to the use of a multimeric major histocompatibility complex (MHC) or a composition according to the disclosure in the development of vaccines.

Another aspect relates to the use of a multimeric major histocompatibility complex (MHC) or a composition according to the disclosure for the identification of epitopes.

In sum, the advantages of the present disclosure include, without limitation, the possibility for detection of multiple (potentially, but exclusively, ><NUM>) different antigen responsive T cells in a single sample. The technology can be used, but is not restricted, for T-cell epitope mapping, immune-recognition discovery, diagnostics tests and measuring immune reactivity after vaccination or immune-related therapies.

This level of complexity allow us to move from model antigens to determination of epitope-specific immune reactivity covering full organisms, viral genomes, cancer genomes, all vaccine components etc. It can be modified in a personalized fashion dependent of the individuals MHC expression and it can be used to follow immune related diseases, such as diabetes, rheumatoid arthritis or similar.

Biological materials are for instance analyzed to monitor naturally occurring immune responses, such as those that can occur upon infection or cancer. In addition, biological materials are analyzed for the effect of immunotherapeutics including vaccines on immune responses. Immunotherapeutics as used here is defined as active components in medical interventions that aim to enhance, suppress or modify immune responses, including vaccines, non-specific immune stimulants, immunosuppressives, cell-based immunotherapeutics and combinations thereof.

The disclosure can be used for, but is not restricted to, the development of diagnostic kits, where a fingerprint of immune response associated to the given disease can be determined in any biological specimen. Such diagnostic kits can be used to determining exposure to bacterial or viral infections or autoimmune diseases, e.g., but not exclusively related to tuberculosis, influenza and diabetes. Similar approach can be used for immune-therapeutics where immune-responsiveness may serve as a biomarker for therapeutic response. Analyses with a barcode labelled MHC multimer library allow for high-throughput assessment of large numbers of antigen responsive T cells in a single sample.

Furthermore, barcode labelled MHC-multimers can be used in combination with single-cell sorting and TCR sequencing, where the specificity of the TCR can be determined by the co-attached barcode. This will enable us to identify TCR specificity for potentially <NUM>+ different antigen responsive T-cells in parallel from the same sample, and match the TCR sequence to the antigen specificity. The future potential of this technology relates to the ability to predict antigen responsiveness based on the TCR sequence. This would be highly interesting as changes in TCR usage has been associated to immune therapy (<NUM>,<NUM>).

Further, there is a growing need for the identification of TCRs responsible for target-cell recognition (e.g., but not exclusive, in relation of cancer recognition). TCRs have been successfully used in the treatment of cancer (<NUM>), and this line of clinical initiatives will be further expanded in the future. The complexity of the barcode labeled MHC multimer libraries will allow for personalized selection of relevant TCRs in a given individual.

Due to the barcode-sequence readout, the barcode labeled MHC multimer technology allow for the interaction of several different peptide-MHC complexes on a single cell surface, while still maintaining a useful readout. When one T-cell binds multiple different peptide-MHC complexes in the library, there relative contribution to T-cell binding can be determined by the number of reads of the given sequences. Based on this feature it is possible to determine the fine-specificity/consensus sequences of a TCR. Each TCR can potential recognize large numbers of different peptide-MHC complexes, each with different affinity (<NUM>). The importance of such quantitative assessment has increased with clinical used of TCRs and lack of knowledge may have fatal consequences as recently exemplified in a clinical study where cross recognition of a sequence related peptide resulted in fatal heart failure in two cases (<NUM>,<NUM>). Thus, this particular feature for quantitative assessment of TCR binding of peptide-MHC molecules related to the present disclosure, can provide an efficient solution for pre-clinical testing of TCRs aimed for clinical use.

Also related to the above, this allows for determination of antigen responsiveness to libraries of overlapping or to very similar peptides. Something that is not possible with present multiplexing technologies, like the combinatorial encoding principle. This allows for mapping of immune reactivity e.g. to mutation variant of viruses, such as, but nor exclusive, HIV.

In broad embodiment, the present disclosure is the use of barcode labelled MHC multimers for high-throughput assessment of large numbers of antigen responsive T cells in a single sample, the coupling of antigen responsiveness to functional and phenotypical characteristic, to TCR specificity and to determine the quantitative binding of large peptide-MHC libraries to a given TCR.

While the foregoing written description of the disclosure enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment of the disclosure, method, and examples herein.

It should be noted that embodiments and features described in the context of one of the aspects of the present disclosure also apply to the other aspects of the disclosure.

<FIG> shows results that act as proof-of-principle for the claimed disclosure. <FIG>, Flow cytometry data of peripheral blood mononuclear cells (PBMCs) from healthy donors.

PBMCs were stained with CMV specific peptide-MHC multimers coupled to a specific nucleotide-barcode. In addition to CMV peptide-MHC reagents the cells were stained in the presence of negative control reagents i.e. HIV-peptide MHC multimers coupled to another specific barcode label and the additional negative control peptide-MHC reagents (p*) not holding a barcode - all multimers were additionally labeled with a PE-fluorescence label. The amounts of MHC multimers used for staining of PBMCs were equivalent to the required amount for staining of <NUM> different peptide-MHC specificities i.e. 1x oligo-labeled CMV specific MHC multimers, 1x oligo-labeled HIV specific MHC multimers and 998x non-labeled p*MHC multimers, so as to give an impression whether background staining will interfere with the true positive signal. Prolonged washing steps were included (either <NUM> (A), <NUM> (B) or <NUM> (C)) after removing the MHC multimers, and data from all experiments are shown. The PE-MHC-multimer positive cells were sorted by fluorescence activated cell sorting (FACS)
<FIG>, Cross threshold (Ct) values from multiplex qPCR of the sorted PE-MHC-multimer positive cells. QPCR was used to assess the feasibility of detecting certain cell specificity through barcode-labeled peptide-MHC-multimers. Reagents associated with a positive control (CMV) barcode and a negative control (HIV) barcode were present during staining, but negative control (HIV) barcode-peptide-MHC multimers should be washed out.

Results shows Ct value only detectable to the CMV peptide-MHC multimer associated barcode, whereas the HIV-peptide MHC multimer associated barcode was not detected.

This experiment is a representative example of several similar experiment performed with other antigen specificities. Overall these data show that it is feasible to.

Together these (and similar data available) provide proof of feasibility for the steps described in <FIG>, <FIG>, and <FIG>.

In i) it is shown that DNA oligos are stable during handling in PBMC's and in blood for a time that will allow staining, washing and isolation of T cells and subsequent amplification of DNA tags.

In ii) Show that a model system consisting of DNA-tagged Dextramers with MHC specificities for CMV, Flu and negative control peptide will locate to and can be captured/sorted with relevant T cell specificities and can be identified by PCR amplification and/or sequencing.

DNA tag oligo design. <NUM>-nucleotide long, biotinylated TestOligo consisting of <NUM>'primer region (22nt yellow)-random barcode region (6xN-nt)-kodon region (<NUM> nt green/underlined)-<NUM>'primer region (20nt blue) were prepared:.

'b' = Biotin-TEG <NUM>' modification
'h' = HEG (terminal modifications).

TestOligo-<NUM>
<IMG>
TestOligo-<NUM>
<IMG>
TestOligo-<NUM>
<IMG>
TestOligo-<NUM>
<IMG>
TestOligo-<NUM>
<IMG>
TestOligo-<NUM>
<IMG>.

Q-PCR probes for quantifying the amount of TestOligos <NUM>-<NUM>:.

The stability of oligo-tags by Q-PCR was analyzed under conditions relevant for T cell isolation:
The testOligos <NUM>-<NUM> were incubated in anticoagulated EDTA blood, and following incubation the amount of each of the testOligos was determined using Q-PCR using the abovementioned primers and probes. The oligo tags were quantified by QPCR with SYBR® Green JumpStart™ Taq ReadyMix™ according to manufacturer's protocol in combination with any capillary QPCR instruments (e.g. Roche LightCycler or Agilent Mx3005P).

Because of the different termini of the testOligos <NUM>-<NUM>, this also was a test of the stability of non-modified DNA oligo tag vs HEG modified <NUM>' and HEG modified <NUM>' and <NUM>' (TestOligo-<NUM>, -<NUM> and -<NUM> respectively).

The results are shown in <FIG>. It is concluded that the stability of the testOligos is appropriately high for all variants tested, to perform the disclosure.

This experiment involves the generation of <NUM> DNA- tagged Dextramers, each with a unique specificity, as follows:.

Each of these Dextramers thus have a unique pMHC specificity (i.e. the three Dextramers have different binding molecules), and each Dextramer carries a unique label (DNA oligonucleotide) specific for that one pMHC specificity.

The library of DNA-tagged Dextramers are screened in a preparation of lymphoid cells such as anticoagulated EDTA blood or preparations of peripheral blood mononucleated cells (PBMC's). Those Dextramers that bind to cells of the cell sample will be relatively more enriched than those that do not bind.

Finally, the MHC/antigen specificity of the enriched Dextramers is revealed by identification of their DNA tags by Q-PCR with DNA tag-specific probes or by sequencing of the DNA tags.

This is an example where the Sample was blood from one CMV positive and HIV negative donor which was modified to generate Peripheral blood mononuclear cells (PBMCs). The Backbone was a dextran conjugate with streptavidin and fluorochrome (Dextramer backbone from Immudex).

The MHC molecules were peptide-MHC (pMHC) complexes displaying either CMV (positive antigen) or HIV (negative antigen) derived peptide-antigens. The MHC molecules were modified by biotinylation to provide a biotin capture-tag on the MHC molecule. The MHC molecule was purified by HPLC and quality controlled in terms of the formation of functional pMHC multimers for staining of a control T-cell population.

The oligonucleotide labels were synthetized by DNA Technology A/S (Denmark). The label was synthetically modified with a terminal biotin capture-tag. The labels were combined oligonucleotide label arising by annealing an A oligonucleotide (modified with biotin) to a partially complimentary B oligonucleotide label followed by enzymatic DNA polymerase extension of Oligo A and Oligo B to create a fully double stranded label. The MHC molecule was synthetized by attaching MHC molecules in the form of biotinylated pMHC and labels in the form of biotin-modified oligonucleotide onto a streptavidin-modified dextran backbone. The MHC molecule further contained a modification (5b) in the form of a fluorochrome. Two different MHC molecules were generated wherein the two individual MHC molecules containing different pMHC were encoded by corresponding individual oligonucleotide labels.

An amount of sample, PBMC's (1b) was incubated with an amount of mixed MHC molecules (<NUM>) under conditions (6c) that allowed binding of MHC molecules to T cells in the sample.

The cell-bound MHC molecules were separated from the non-cell bound MHC molecules (<NUM>) by first a few rounds of washing the PBMC's through centrifugation sedimentation of cells and resuspension in wash buffer followed by Fluorescence Activated Cell Sorting (FACS) of fluorochrome labeled cells. T cells that can efficiently bind MHC molecules will fluoresce because of the fluorochrome comprised within the MHC molecules; T cells that cannot bind MHC molecules will not fluoresce. FACS-sorting leads to enrichment of fluorescent cells, and hence, enrichment of the MHC molecules that bind T cells of the PBMC sample.

FACS isolated cells were subjected to quantitative PCR analysis of the oligonucleotide label associated with the MHC molecules bound to the isolated cells to reveal the identity of MHC molecules that bound to the T cells present in the sample.

This experiment thus reveal the presence of T cells in the blood expressing a T cell receptor that recognize/binds to peptide-MHC molecules comprised in the peptide-MHC multimeric library.

After sorting and qPCR the resultant Ct values confirmed that Labels were successfully recovered and enriched only when associated with the CMV epitope, while they were not detected when associated with the HIV epitope (<FIG>).

Thus, it was verified that the 2OS labels were recovered after cellular interaction, sorting and qPCR only T cell recognizing the given pMHC molecule were present in the sample.

Detection of a B7 CMV pp65 TPR specificity amongst negative control barcoded pMHC dextramers. A unique 2OS barcode was associated with the positive control reagents in <NUM>. , while another unique 2OS barcode was associated with the positive control reagents in <NUM>. The spare barcode in each experiment was associated with the HIV negative control reagent. A, Representative dot plot showing the PE positive population after staining with the CMV and HIV pMHC multimers carrying separate 2OS-barcodes. B, Ct values from multiplex qPCR of the sorted PE-pMHC-dextramer positive cells. Cells were stained with <NUM>. respectively. Reagents associated with a positive control (CMV) 2OS barcode and a negative control (HIV) 2OS barcode were present during staining, but the negative control (HIV) barcoded pMHC dextramer was evidently washed out. The results obtained from two individual experiments are presented in separate bars. Approximately <NUM> cells were applied in each separate PCR. QPCR was run in duplicates and Ct values are shown as mean ±range of duplicates.

This is an example where the Sample (<NUM>) was blood from one CMV positive and HIV negative donor which was modified (1b) to generate Peripheral blood mononuclear cells (PBMCs).

The Backbone (<NUM>) was a dextran conjugate with streptavidin and fluorochrome (Dextramer backbone from Immudex).

The example is similar to example <NUM> except that a <NUM> fold excess of MHC molecules with irrelevant MHC molecules but without label were included. The MHC molecules used (<NUM>) are peptide-MHC (pMHC) complexes displaying either CMV (positive antigen) or HIV (negative antigen) derived peptide-antigens or pMHC complexes displaying irrelevant peptide antigen. The MHC molecules were modified (3b) by biotinylation to provide a biotin capture-tag on the MHC molecule. The MHC molecules were purified (2c) by HPLC. The Labels (<NUM>) were oligonucleotides. The oligonucleotides were synthetized (4a) by DNA Technology A/S (Denmark). The labels were synthetically modified (4b) with a terminal biotin capture-tag.

The MHC molecule (<NUM>) was synthetized (5a) by attaching MHC molecules in the form of biotinylated pMHC and labels in the form of biotin-modified oligonucleotide onto a streptavidin-modified dextran backbone. The MHC molecule further contained a modification (5b) in the form of a fluorochrome. Three different MHC molecules were generated wherein the two of these individual MHC molecules containing CMV- and HIV-directed pMHC were encoded for by corresponding individual oligonucleotide labels. MHC molecules with irrelevant MHC molecules were not encoded for with oligonucleotide label. An amount of sample, PBMC's (1b) was incubated with an amount of mixed MHC molecules (<NUM>) in a ratio of <NUM>:<NUM> and in addition a <NUM> fold of unlabeled p*MHC labeled backbone was included under conditions (6c) that allowed binding of MHC molecules to T cells in the sample.

This experiment thus revealed the peptide-MHC specificity of the T cell receptors of the T cells present in the blood sample. It further revealed the feasibility of enriching for T cells specific for the CMV-antigen (positive) over the HIV-antigen (negative) and an excess of MHC molecule displaying irrelevant peptide antigens.

After sorting and qPCR the resultant Ct values confirmed that Labels were successfully recovered and enriched for only when associated with the CMV epitope, while they were not detected when associated with the HIV epitope (<FIG>).

It was verified that the 2OS labels were recovered after cellular interaction, sorting and qPCR only if they were associated with positive control reagents.

Detection of a CMV specificity amongst negative control barcoded pMHC dextramers. A unique barcode is associated with the positive control reagents in <NUM>. , while another unique barcode is associated with the positive control reagents in <NUM>. The spare barcode in each experiment is associated with the HIV negative control reagent. In addition 998x unlabeled negative control reagents are present in both <NUM>.

A, Ct values from multiplex qPCR of the sorted PE-pMHC-dextramer positive cells. Cells were stained with <NUM>. respectively. Reagents associated with a positive control (CMV) barcode and a negative control (HIV) barcode were present during staining, but the negative control (HIV) barcoded pMHC dextramer was evidently washed out. Approximatly <NUM> cells were analyzed in each separate qPCR. The estimated number of barcodes bound per cell relative to the obtained Ct-values. It is evident that there are some differences in the Ct values shown in B, even though the same number of cells were present in all qPCRs. This is however leveled when the values are normalized in respect to their specific probes. QPCR was run in duplicates, here showing mean ± range of dublicates.

This is an example where the Sample (<NUM>) was blood which was modified (1b) to generate Peripheral blood mononuclear cells (PBMCs).

The MHC molecules (<NUM>) are peptide-MHC (pMHC) complexes displaying an <NUM>-<NUM> amino acid peptide-antigen. The MHC molecule was modified (3b) by biotinylation to provide a biotin capture-tag on the MHC molecule. The MHC molecule was purified (2c) by HPLC. The Label (<NUM>) was an oligonucleotide. The oligonucleotide label was synthetized (4a) by DNA Technology A/S (Denmark) and was synthetically modified (4b) with a terminal biotin capture-tag. In parts of the example the oligonucleotide label was further modified by annealing to a partially complimentary oligonucleotide label giving rise to a combined oligonucleotide label.

The MHC molecule (<NUM>) was synthetized (5a) by attaching MHC molecules in the form of a biotinylated pMHC and labels in the form of a biotin-modified oligonucleotide onto a streptavidin-modified dextran backbone (Dextramer backbone from Immudex, Denmark). The MHC molecule further contains a modification (5b) in the form of a fluorochrome. A library of <NUM> different MHC molecules were generated wherein individual MHC molecules containing different pMHC were encoded by corresponding individual oligonucleotide labels.

An amount of sample, PBMC's (1b) was incubated with an amount of a library of MHC molecules (<NUM>) under conditions (6c) (e.g. incubation time, buffer, pH and temperature) allowing binding of MHC molecules to T cells in the sample.

FACS isolated cells were subjected to PCR amplification of the oligonucleotide label associated with the MHC molecules bound to cells. Subsequent sequencing of individual DNA fragments generated by the PCR reaction revealed the identity of MHC molecules that bound to the T cells present in the sample.

This experiment thus revealed the peptide-MHC specificity of the T cell receptors of the T cells present in the blood sample.

This example shows the feasibility for detection of antigen responsive T-cell in a large mixture of different pMHC multimer (MHC molecules). We show the sensitivity of the barcode-labelled MHC multimers being at least able to detect <NUM>% of specific T-cell out of CD8 T cells. We find exact correlation withprevious described (low throughput) methods.

Schematic presentations of the number of specific 1OS barcode reads mapped to seven different samples. A <NUM>% B7 CMV pp65 TPR response (barcode <NUM>) were spiked into a HLA-B7 negative BC in fivefold dilutions, creating seven samples (<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%). This BC has a population of A11 EBV-EBNA4 specific T cell (corresponding to barcode <NUM>). Samples were stained with the same panel comprising <NUM> differently 1OS barcoded-pMHC-dextramers. The bars show the total reads normalized to the input panel in each sample. Experiments were performed in duplicate. Here showing mean.

Schematic presentations of the number of specific 2OS barcode reads mapped to seven different samples. A <NUM>% B7 CMV pp65 TPR response (barcode A3B18) were spiked into a HLA-B7 negative BC in fivefold dilutions, creating seven samples (<NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% and <NUM>%). This BC has a population of A11 EBV-EBNA4 specific T cell (corresponding to barcode A1B4). Samples were stained with the same panel comprising <NUM> differently 2OS barcoded-pMHC-dextramers. The bars show the total reads normalized to the input panel in each sample. Experiments were performed in duplicate. Here showing mean.

Examples <NUM> is conducted exactly as examples <NUM>, with the only difference that we have used a different sample. Here we detect antigen responsive T-cells in <NUM> different donor blood samples.

This example shows the feasibility to detect numerous different specificities in different donor samples using DNA barcode labelled MHC multimers. Obtained data show the feasibility for high-throughput screening of T-cell reactivity in numerous donor to assess immune reactivity associated with disease development, vaccination, infection etc..

A schematic presentations of the number of specific 1OS barcode reads mapped to six different samples. Six BCs were stained with the same panel comprising <NUM> differently 1OS barcoded-pMHC-dextramers. Bar charts show the total reads normalized to the input panel in each sample (p<<NUM>). Each pie chart show significant (p<<NUM>) reads mapped to that sample.

Schematic presentations of the number of specific 2OS barcode reads mapped to six different samples. Six BCs were stained with the same panel comprising <NUM> differently 2OS barcoded-pMHC-dextramers. Bar charts show the total reads normalized to the input panel in each sample (p<<NUM>).

Claim 1:
A method for detecting antigen responsive T cells in a sample comprising:
i) providing a composition comprising different subsets of multimeric MHCs,
wherein each multimeric MHC comprises i) two or more given MHC-peptide molecules linked to a backbone molecule, and ii) at least one nucleic acid molecule linked to said backbone molecule, wherein said at least one nucleic acid molecule comprises a <NUM>' first primer region, a central stretch of nucleic acids (barcode region) and a <NUM>' second primer region, wherein said barcode region serves as a specific label for said two or more given MHC-peptide molecules;
wherein each subset of multimeric MHC has a unique barcode region specific for the given MHC-peptide molecule; and
ii) contacting said multimeric MHCs with said sample; and
iii) removing unbound multimeric MHCs before amplification by washing and spinning; and
iv) detecting binding of the multimeric MHCs to said antigen responsive T cells, thereby detecting T cells responsive to an antigen present in said multimeric MHCs,
wherein said binding of said multimeric MHCs to said antigen responsive T cells is detected by
a) amplifying by PCR the barcode region of said nucleic acid molecules linked to the cell-bound multimeric MHCs , and
b) sequencing of said amplified barcode regions.