US20170233807A1

US20170233807A1 - Epigenetic Markers for the Identification of Blood Sub-cells of Type 1

Info

Publication number: US20170233807A1
Application number: US15/477,768
Authority: US
Inventors: Sven Olek; Ivana TÜRBACHOVA - deceased
Original assignee: Epiontis GmbH
Current assignee: Precision for Medicine GmbH
Priority date: 2008-12-16
Filing date: 2017-04-03
Publication date: 2017-08-17
Also published as: US20190241957A1

Abstract

The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD3a/b/c/d/g genes, in particular their “upstream” regulatory regions, and in particular the promoter and other conserved regions of the gene cd3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4 or/and memory and/or naïve T lymphocytes. Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3a/b/c/d for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.

Description

CROSS REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part application of co-pending application Ser. No. 13/139,808, filed Oct. 18, 2011; which is a National Stage Application of International Application Number PCT/EP2009/008764, filed Dec. 8, 2009; which claims priority to European Application No. 08021838.1, filed Dec. 16, 2008; all of which are incorporated herein by reference in their entirety.
The Sequence Listing for this application is labeled “SeqList-14Aprl7-ST25.txt”, which was created on Apr. 14, 2017, and is 54 KB. The entire contents are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

T-lymphocytes are a major component of the mammalian immune system. Both CD4 and CD8 T-cells are responsible for proper functioning of said immune system. Whereas CD8 T-cells mediate the cytotoxic immune defence, CD4 cells—the so called helper T-cells—assist both the humoral and the cell mediated immune defence. The heterodimeric T-cell antigen receptor (TCR) is bound to a monomorphic protein complex called CD3. CD3 consists of sub-elements CD3 γ, -δ, and -ε, and is expressed on all peripheral T-cells, but only on a subset of the thymocytes. The genes for CD3 γ, -δ, and -ε are encoded on neighbouring loci on chromosom 11 (11q23).
Role of CD3 in signal transduction—The function of the CD3-chains is both the formation and the transport of the full CD3 complex to the cell surface, as well as the signal transduction upon stimulation through the heterodimeric T-cell antigen receptor. The amino acid sequence of the cytoplasmatic part of CD3γ, -δ, -ε each contains a motif that becomes phosphorylated upon stimulation, and thus activated. This so called ITAM (immunoreceptor tyrosine-based activated motif) serves as docking point for different tyrosine kinases. Individuals with defective CD3γ or-ε-chains suffer from a severe clinical autoimmune deficiency.
Even though almost all cells in an individual contain the exact same complement of DNA code, higher organisms must impose and maintain different patterns of gene expression in the various types of tissue. Most gene regulation is transitory, depending on the current state of the cell and changes in external stimuli. Persistent regulation, on the other hand, is a primary role of epigenetics—heritable regulatory patterns that do not alter the basic genetic coding of the DNA. DNA methylation is the archetypical form of epigenetic regulation; it serves as the stable memory for cells and performs a crucial role in maintaining the long-term identity of various cell types.
The primary target of methylation is the two-nucleotide sequence Cytosine-Guanine (a ‘CpG site’); within this context cytosine (C) can undergo a simple chemical modification to become 5-methyl-cytosine. In the human genome, the CG sequence is much rarer than expected, except in certain relatively dense clusters called ‘CpG islands’. CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA 90: 11995-9, 1993).
Aberrant methylation of DNA is frequently associated with the transformation from healthy to cancerous cells. Among the observed effects are genome-wide hypomethylation, increased methylation of tumour suppressor genes, and hypomethylation of many oncogenes (reviewed, for example, by Jones and Laird, Nature Genetics 21:163-167, 1999; Esteller, Oncogene 21:5427-5440, 2002; and Laird, Nature Reviews/Cancer 3:253-266, 2003). Methylation profiles have been recognized to be tumour specific (i.e., changes in the methylation pattern of particular genes or even individual CpGs are diagnostic of particular tumour types), and there is now an extensive collection of diagnostic markers for bladder, breast, colon, oesophagus, stomach, liver, lung, and prostate cancers (summarized, for example, by Laird, Nature Reviews/Cancer 3:253-266, 2003).
Flanagan et al. (in Flanagan B F, Wotton D, Tuck-Wah S, Owen M J. DNase hypersensitivity and methylation of the human CD3G and D genes during T-cell development. Immunogenetics. 1990; 31(1):13-20.) describe that the mouse and human CD3G and D genes are organized in opposite transcriptional orientation, their 5′ ends being separated by about 1.6 kilobases (kb) of DNA. The molecular basis of the tissue-specific regulation of expression of the human CD3G and D genes were examined using DNase I hypersensitivity and CpG methylation analysis in the mouse. The authors try to define areas 3′ to the D gene and within the intergenic region which contain regulatory elements that influence both CD3D and G expression and show that transcription from the CD3D and G genes may occur initially from a methylated promoter. Significantly, the 3′ regulatory region was shown to adopt an open chromatin structure prior to lineage commitment and before CD3 transcription. The quite limited enzymatic analysis of Flanagan is based on a region in the mouse that has no homology to any region as found in the human. Furthermore, the paper does not identify the regions as analyzed as suitable for the identification of DC3 lymphocytes.
Clevers et al. (in Clevers H, Lonberg N, Dunlap S, Lacy E, Terhorst C. An enhancer located in a CpG-island 3′ to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter. EMBO J. 1989 September; 8(9):2527-35.) describe that the gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (−228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive. In contrast to the preferred embodiment of the present invention, Clevers et al. analyze a remotely located enhancer region of 3′ to the TCR/CD3-epsilon gene.
Hamerman et al. (in: Hamerman J A, Page S T, Pullen A M. Distinct methylation states of the CD8 beta gene in peripheral T cells and intraepithelial lymphocytes. J Immunol. 1997 Aug. 1; 159(3):1240-6) distinguish between CD4 and CD8 T-lymphocytes.
EP 1 213 360 describes a method of identifying a cell, tissue or nucleus, comprising collecting information on the methylation pattern of DNA isolated from the cell, tissue or nucleus and analyzing the resultant information.
WO 2004/050706 describes a sub-group of T-cells, and relates to characteristics of regulatory T-cells which define them as such. The application also describes the uses of such T-cells, compositions comprising them, and chemokines which recruit them in the modulation of an immune response.
Finally, EP 1 826 279 describes a method, in particular an in vitro method, for identifying FoxP3-positive regulatory T cells, preferably CD25⁺ CD4⁺ regulatory T cells of a mammal, comprising analyzing the methylation status of at least one CpG position in the gene foxp3 or an orthologous or paralogous gene thereof, and the use of DNA-methylation analysis of the gene of the transcription factor FoxP3 for a detection and quality assurance and control of regulatory T cells.
While the measurement and determination of CD4 and CD8 cells is generally easy and is usually achieved through analyzing the expression of said antigens on the cellular surface, clinically, it remains challenging to determine these cell types, since for the commonly used FACS analysis the cell samples need to be freshly isolated or immediately fixated in order to keep the cell entities intact. Thus, the detection of T lymphocytes, while desirous, is problematic, particularly for routine applications.
In view of the above, it is an object of the present invention to provide an improved and in particular robust method based on DNA methylation analysis as a superior tool in order to more conveniently and reliably identify T-lymphocytes.

BRIEF SUMMARY

The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 and/or CD3CD8 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD38/γ/ε genes, in particular their “upstream” regulatory regions, and in particular the promoter and other conserved regions of the gene for CD3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4⁺ T lymphocytes and memory and naive CD8⁺ T lymphocytes. The present invention is further related at analyzing the methylation status of at least one CpG position in the genes SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK which also allows the unambiguously identification of all CD3 positive T lymphocytes. For further unambiguous identification of all CD8 cells, also the equivalent analysis GNGT2, CRTAM, IL2RB and ZBTB32 can be employed. Among the CD3 positive T lymphocytes, these markers are capable to segregate between CD8 and CD4 positive cells. Equivalently, FLJ00060, FLJ38379, PPP6C, CD226, ZBTB7B and TNFAIP8 are capable of positively identifying CD4 expressing cells in whole blood and segregate between CD4 and CD8 positive CD3 positive cells.
Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3χ/δ/ε or SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK or GNGT2, CRTAM, IL2RB and ZBTB32. or FLJ00060, FLJ38379, PPP6C, CD226, ZBTB7B and TNFAIP8 for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples. It is one aim of this invention to provide a novel, more robust means to quantitatively detect and measure particular subsets of the blood within any solid organs or any body fluid of a mammal.
Employing this method, the inventors provide for novel, not previously known means of determining, quantitating and routinely measuring T lymphocytes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the differentially methylated gene region as found for CD3 g and d and are indicated by thick lines as “blast hits”.

FIGS. 2A-2B show the analysis of amplicon No. 1405 (SEQ ID NO: 6), bisulfite strand 2, sequencing direction: forward. Relevant positions are indicated in bold.

FIGS. 3A-3B show the analysis of amplicon No. 1406, bisulfite strand 2, sequencing direction: reverse. Relevant positions are indicated in bold.

FIGS. 4A-4B show the analysis of amplicon No. 1406 (SEQ ID NO: 7), bisulfite strand: 2, sequencing direction: forward. Relevant positions are indicated in bold.

FIGS. 5A-5B show the analysis of amplicon No. 1408 (SEQ ID NO: 8), bisulfite strand: 2, sequencing direction: forward. Relevant positions are indicated in bold.

FIGS. 6A-6E show the regulatory regions of the γ,δ-T-cell receptor, including regions Nr. 1405 1406 und 1408 on chromosome 11 (NCBI36:11:117714000:117730500:1). Exon sequences are underlined for CD3 δ, and double underlined for CD3 γ. CGs are in bold.

FIG. 7 shows an example of bisulfite-treated nucleotide sequence having non-methylated CpG motifs.

FIG. 8 shows an example of a bisulfite-treated nucleotide sequence having methylated CpG motifs.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the sequence of the region as considered to have a specific methylation pattern in CD3 cells.
SEQ ID NOs: 2 to 5 and 9 show the sequences of oligonucleotides used in the Illumina Chip-Fragment assay for CD3γ and CD3δ and CD3ε.
SEQ ID NOs: 6 to 8 show the reference sequences of amplicon No. 1405 (SEQ ID NO: 6), amplicon No. 1406 (SEQ ID NO: 7), and amplicon No. 1408 (SEQ ID NO: 8).
SEQ ID NOs: 10 to 38 show the sequences around the CpG positions as analyzed in other preferred T-lymphocyte markers of the present invention according to example 2.
SEQ ID NOs: 39 to 65 show the sequences as depicted in the alignments of FIGS. 2A to 5B. If not stated otherwise, the sequences are ordered in the succession of their appearance in FIGS. 2A to 5B.
SEQ ID NOs: 68 and 69 show the sequences of the reverse complement sequences of SEQ ID NO: 5 and SEQ ID NO: 2, which reverse complementary sequences are used to amplify an amplicon from nucleic acid molecules comprising SEQ ID NO: 1.
SEQ ID NOs: 70 and 71 show the sequences of a primer pair used to amplify one strand of a bisulfite-treated nucleic acid molecule comprising SEQ ID NO: 1
SEQ ID NOs: 72 and 73 show the sequences of a primer pair used to amplify the complementary strand of the bisulfite-treated nucleic acid molecule comprising SEQ ID NO: 1.
SEQ ID NO: 74 shows the sequence of a portion of the bisulfite-treated nucleic acid sequence of SEQ ID NO: 1 in which the CpG motifs are non-methylated, the shown sequence comprising the preferred sequences of SEQ ID NOs: 72, 73, 76, 77, and 78.
SEQ ID NO: 75 shows the sequence of a portion of the bisulfite-treated nucleic acid sequence of SEQ ID NO: 1 in which the CpG motifs are methylated, the shown sequence comprising the preferred sequences of SEQ ID NOs: 79-81.
SEQ ID NO: 76 shows the forward primer used for amplification of a portion of the bisulfite-treated SEQ ID NO: 1 having non-methylated CpGs.
SEQ ID NO: 77 shows the reverse primer used for amplification of a portion of the bisulfite-treated SEQ ID NO: 1 having non-methylated CpGs.
SEQ ID NO: 78 shows the sequence of a probe used for quantitative PCR amplification of a portion of the bisulfite-treated SEQ ID NO:1 with primers of SEQ ID NOs: 76 and 77.
SEQ ID NO: 79 shows the forward primer used for amplification of a portion of the bisulfite-treated SEQ ID NO: 1 having methylated CpGs.
SEQ ID NO: 80 shows the reverse primer used for amplification of a portion of the bisulfite-treated SEQ ID NO: 1 having methylated CpGs.
SEQ ID NO: 81 shows the sequence of a probe used for quantitative PCR amplification of a portion of the bisulfite-treated SEQ ID NO: 1 with primers of SEQ ID NOs: 79 and 80.

DETAILED DESCRIPTION

The present invention solves the above object by providing a method for identifying T-lymphocytes in a mammal, in particular in a sample derived from a mammal, comprising analysing the methylation status of at least one CpG position in one or more of the genes for CD3 γ, -δ, and -ε, wherein a demethylation of at least one CpG position to at least 90% in said sample is indicative for a CD3⁺ T-lymphocyte cell, in particular a CD3⁺ CD4⁺, and/or CD3⁺ CD8⁺ T-lymphocyte cell.
The present invention is based on the surprising finding of the inventors that the identification of CD3 gene as a specific epigenetic marker can greatly facilitate the clinical routine application of the analysis of the above markers. In contrast to FACS and mRNA measurements, the respective measurement(s) can be done independent of purification, storage and to quite some extend also to tissue quality.
In another preferred embodiment of the method according to the present invention, said at least one CpG position in said sample is demethylated to more than 91% and preferably more than 92% and most preferred more than 95%.
This concept is based on specific demethylation of the CD3 regions in CD3 positive T-lymphocytes. Using a simple and precise quantitative PCR method, the inventors show that CD3 demethylation represents a surrogate marker for T-lymphocyte counts in blood or tissues. The present inventors have thus identified particular regions within the CD3 gene, which are functionally involved in, or mandatory associated with, the existence of CD3 positive T-lymphocytes. In one preferred embodiment one very good region is either the promoter or the TLSDR with e.g. the nucleotide sequence according to SEQ ID NO: 1 and others, containing many CpG motifs, which display a differential methylation status when cells expressing CD3 in either CD4⁺ or CD8⁺ cells compared with all other cells, not expressing CD3 if, for example, the bisulphite sequencing method is used. An example of bisulfite-treated nucleotide sequence according to SEQ ID NO: 1 having non-methylated CpG motifs is provided in SEQ ID NO: 74 and FIG. 7. A further example of the similar bisulfite-treated nucleotide sequence according to SEQ ID NO: 1 having methylated CpG motifs is provided in SEQ ID NO: 75 and FIG. 8.
The inventors could demonstrate that in CD3⁺ cells the CpG motifs are almost completely demethylated (i.e. to more than 70%, preferably 80%, preferably, more than 90% and most preferred more than 95%), whereas the same motifs are completely methylated in all CD3⁻ cells. The differential methylation of the CpG motifs within the aforementioned region correlates with CD3 expression. Thus, determination of the methylation status of the CD3 locus could become a valuable tool to identify T-lymphocytes, such as will be required/or at least of some value for measuring T-lymphocytes in autoimmune diseases, transplant rejections, cancer, allergy, or just the T-lymphocytes related immune status in any envisionable context, when desired. The assays allows measurement of T-lymphocytes without purification or any staining procedures. It even reports in solid tumors or other solid tissues the number of cells demethylated in said region, thus showing the total amount of CD3 positive tumor infiltrating T-lymphocytes.
The inventors have shown that the potential for constitutive expression of CD3 in T-lymphocytes coincides with epigenetic, i.e., DNA methylation based regulation. DNA methylation is a biologically and chemically stable epigenetic modification, resulting in long-term gene expression changes. The inventors found demethylation at the human CD3 locus to be restricted to T-lymphocytes when tested against all major peripheral blood cell types and a selection of non-blood cells. These data indicated that epigenetic modifications in the CD3 locus serve as valuable marker for the identification of cells with the phenotype of T-lymphocyte, regardless of the expression of the specific delta or gamma sub-chains.
In another preferred aspect of the method according to the present invention the methylation status of at least one CpG position in the genes SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK is analysed in analogy to what is described herein for CD3. These genes thus also allow the unambiguous identification of all CD3 positive T lymphocytes. Thus, in a preferred embodiment of the method according to the present invention, said at least one CpG position is present in the 5′ region upstream from the transcription start, promoter region, intron, and/or exon/intron border within the gene(s) SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK.
In order to further unambiguously identify all CD8 cells, the present invention in another preferred aspect thereof provides the equivalent analysis of the genes GNGT2, CRTAM, IL2RB and ZBTB32 among the CD3 positive T lymphocytes, as these markers are capable to segregate between CD8 and CD4 positive cells. This analysis is preferably 5 performed simultaneously or subsequently to the analysis for the CD3 phenotype of the T-lymphocytes.
Equivalently, FLJ00060, FLJ38379, PPP6C, CD226, ZBTB7B and TNFAIP8 are capable of positively identifying CD4 expressing cells in whole blood and segregate between CD4 and CD8 positive CD3 positive cells.
Another preferred aspect of the method according to the present invention is directed at the use of DNA-methylation analysis of the genes CD3γ/δ/ε, or SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK or GNGT2, CRTAM, IL2RB and ZBTB32 or FLJ00060, FLJ38379, PPP6C, CD226, ZBTB7B and TNFAIP8 for the detection and quality assurance and control of T lymphocytes.
In another preferred embodiment of the method according to the present invention, said at least one CpG position is present in the 5′ region upstream from the transcription start, promoter region, intron, and/or exon/intron border within the CD3 gene. The present invention also provides the surprising finding that in particularly preferred regions of the gene for CD3, the so-called “TLSDRs” (T lymphocyte specific demethylated regions), the CpG motifs are almost completely demethylated (i.e. to more than 90%, preferably 91%, preferably, more than 92% and most preferred more than 95%), whereas the same motifs are completely methylated in all non T lymphocytes. Thus, this region and the diagnostic uses thereof also provide a valuable and reliable tool for a diagnostic analysis according to the present invention. The TLSDR according to the present invention are located in amplicon No. 1405 (SEQ ID NO: 6), amplicon No. 1406 (SEQ ID NO: 7), and/or amplicon No. 1408 (SEQ ID NO: 8). All of these amplicons are parts of the overall region of interest of the present invention as depicted in SEQ ID No. 1. Upon bisulfite treatment of SEQ ID NO:1, a preferred region of the present invention is shown in FIGS. 7 and 8 and is disclosed as sequences of SEQ ID NOs: 74 and 75.
In a preferred embodiment of the method according to the present invention, said analysis of the methylation status comprises amplification with at least one primer of suitable primer pairs that can be suitably designed based on SEQ ID NO: 1, preferably oligomers according to any of SEQ ID NOs: 2 to 5. The person with ordinary skill in the art will readily appreciate that, for example, in order to amplify a sequence of SEQ ID NO: 1 located between oligomers of SEQ ID NO: 5 and SEQ ID NO: 2, primers are designed such that the primers comprise the reverse complement sequences of SEQ ID NOs: 5 and 2. Such reverse complement primers of SEQ ID NOs: 5 and 2 are primer sequences of SEQ ID NO: 68 and 69, respectively. Therefore, in a preferred embodiment primers to amplify an amplicon from SEQ ID NO: 1 are SEQ ID NOs: 68 and 69. SEQ ID NO: 68 is the reverse complement of SEQ ID NO: 5 and SEQ ID NO: 69 is the reverse complement of SEQ ID NO: 2.
In one embodiment of the method according to the present invention, said at least one CpG position is present in the region comprising SEQ ID NOs: 2-5.
In a preferred embodiment, the method comprises bisulfite treating the genomic DNA isolated from a cell and amplifying the region comprising SEQ ID NO: 1 using a primer pair designed to amplify bisulfite-treated SEQ ID NO: 1. The primer pair can be designed based on the bisulfite-treated SEQ ID NO: 1. Methods to design primers to amplify bisulfite-treated genomic DNA are within the knowledge of the art.
For example, a primer pair to amplify one strand of the bisulfite-treated genomic DNA comprising SEQ ID NO: 1 comprises:
i) a forward primer of SEQ ID NO: 70, and
ii) a reverse primer of SEQ ID NO: 71.
A primer pair to amplify the complementary strand of the bisulfite-treated genomic DNA comprising SEQ ID NO: 1 comprises:
(i) a forward primer of SEQ ID NO: 72, and
(ii) a reverse primer of SEQ ID NO: 73.
A preferred embodiment is a bisulfite-treated sequence of SEQ ID NO: 1. A further preferred embodiment is a partial sequence of bisulfite-treated sequence of SEQ ID NO: 1, which partial sequence comprises non-methylated CpG motifs and is SEQ ID NO: 74. A further embodiment is a partial sequence of bisulfite-treated sequence of SEQ ID NO: 1, which partial sequence comprises methylated CpG motifs and is SEQ ID NO: 75.
Preferably, the amplification involves a polymerase enzyme, a PCR or chemical amplification reaction or other amplification methods as known to the person of skill as described below, e.g. in the context of MSP, HeavyMethyl, Scorpion, MS-SNUPE, MethylLight, bisulfite sequencing, methyl specific restriction assays. With the amplification, the amplicon of the TLSDR or any other region in the CD3 gene or any paralog or ortholog as described herein is produced that is a particularly preferred “tool” for performing the method(s) according to the present invention. Consequently, an oligomer according to any of SEQ ID NOs: 2 to 5 or the amplicon as amplified by a primer pair as mentioned above constitute preferred embodiments of the present invention.
The person of skill will furthermore be able to select specific subsets of CpG positions in order to minimise the amount of sites to be analyzed, for example at least one of CpG position 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of the amplicon No. 1405 (SEQ ID NO: 6), amplicon No. 1406 (SEQ ID NO: 7), and amplicon No. 1408 (SEQ ID NO: 8), or all sites as present on the amplicons or according to SEQ ID NO: 1 or other sequences in the CD3 locus. The positions are numerically counted from the 5′-end of the amplicon as generated and analysed. Preferred are combinations of 4, 5, 6, or 7 positions, which are producing enough information in order to be informative in the context of the present invention.
In order to analyze the methylation status of CpG positions, any known method to analyse DNA methylation can be used. In a preferred embodiment of the method according to the present invention, the analysis of the methylation status comprises a method selected from methylation specific enzymatic digests, bisulphite sequencing, analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, Ms-SNuPE or other methods relying on a detection of amplified DNA. These methods are well known to the person of skill, and can be found in the respective literature. For example, primers and a probe to be used according to the instant invention for the detection of non-methylated CpG motifs in the preferred partial nucleotide sequence of bisulfite-treated SEQ ID NO: 1, i.e. SEQ ID NO:74, are SEQ ID NOs: 76-78. Primers and a probe to be used according to the instant invention for the detection of methylated CpG motifs in the preferred partial nucleotide sequence of bisulfite-treated SEQ ID NO:1, i.e. SEQ ID NO:75, are SEQ ID NOs: 79-81.
In a preferred embodiment of the method according to the present invention, said method is suitable for routine application, for example on a DNA-chip. Based on the above information and the respective literature, the person of skill will be able to adjust the method as above to such settings.
In another preferred embodiment of the method according to the present invention, the identification comprises a distinction of said T-lymphocytes from all major peripheral blood cell types or non-blood cells, preferably, but not limited to, CD14, CD15, CD19, and CD56.
In yet another preferred embodiment of the method according to the present invention, the sample is selected from a mammalian body fluid, including human blood samples, or a tissue, organ or cell type blood sample, a sample of blood lymphocytes or a fraction thereof. Preferably, said mammal is a mouse, rat, monkey or human. The samples can be suitably pooled, if required.
Another preferred aspect of the method according to the present invention then further comprises the step of concluding on the immune status of said mammal based on said T-lymphocytes as identified. The general lymphocyte population can be quantified and either be used as a benchmark to relatively quantify further detailed subpopulations such as the CD3CD4CD25 positive regulatory T cells or it can be used to finally detect this population to determine the overall immune activity status.
In yet another preferred embodiment of the methods according to the present invention, the mammal suffers from or is likely to suffer from autoimmune diseases, transplant rejections, cancer, and/or allergy.
Another preferred aspect of the method according to the present invention is related to a method for monitoring the level of CD3⁺ CD4⁺, and/or CD3⁺ CD8⁺ T-lymphocytes in a mammal, comprising a method as above, and comparing the amount of T-lymphocytes as identified to an earlier sample taken from the same mammal, and/or to a control sample. In yet another preferred embodiment of the methods according to the present invention, the mammal suffers from or is likely to suffer from autoimmune diseases, transplant rejections, cancer, and/or allergy.
Another preferred aspect of the method according to the present invention then relates to a method as above, further comprising measuring and/or monitoring the amount of said T-lymphocytes in response to chemical and/or biological substances that are provided to said mammal.
Another preferred aspect of the method according to the present invention relates to an oligomer according to any of SEQ ID NOs: 2 to 5, SEQ ID NOs: 68 to 73, SEQ ID NOs: 76, 77, 79 and 80, or an amplicon as amplified by a primer pair based on SEQ ID NO: 1 and designed as described above, in particular the amplicon No. 1405 (SEQ ID NO: 6), amplicon No. 1406 (SEQ ID NO: 7), and amplicon No. 1408 (SEQ ID NO: 8). A particularly preferred primer pair are SEQ ID NOs: 70 and 71 to amplify one strand of a preferred partial sequence of SEQ ID NO: 1. Another particularly preferred primer pair are SEQ ID NOs:72 and 73 to amplify the complementary strand of the preferred partial sequence of SEQ ID NO:1. A further preferred primer pair are SEQ ID NOs: 79 and 80 to be used with a probe of SEQ ID NO: 81 for quantitative PCR of a preferred partial sequence of bisulfite-treated SEQ ID NO: 1. An additional primer pair are SEQ ID NOs: 76 and 77 to be used with a probe of SEQ ID NO: 78 for quantitative PCR of a preferred partial sequence of bisulfite-treated SEQ ID NO: 1.
Yet another preferred aspect of the present invention then relates to a kit for identifying and/or monitoring CD3⁺ CD4⁺, and/or CD3⁺ CD8⁺T-lymphocytes in a mammal 5 based on the analysis of the methylation status of CpG positions in the gene CD3, comprising materials for performing a method according the present invention as described herein. Preferably, said kit comprises a) a bisulfite reagent, and b) materials for the methylation analysis of CpG positions as comprised by the amplicon No. 1405 (SEQ ID NO. 6), amplicon No. 1406 (SEQ ID NO: 7), and amplicon No. 1408 (SEQ ID NO: 8). Further preferred, the 10 positions consist of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of said amplicons. Most preferred are 5, 6, or 7 or all positions on said amplicon.
Finally, the present invention also encompasses the use of an oligomer or amplicon or a kit according to the present invention for identifying and/or for monitoring CD3⁺ CD4⁺, and/or CD3⁺ CD8⁺ T-lymphocytes in a mammal.
In summary, using the CD3 marker, the inventors very specifically identified (but not differentiated) both CD3/CD4 positive as well as CD3/CD8 positive T lymphocytes. Using the marker CD8beta, for example CD8 positive lymphocytes could then be distinguished from CD4 lymphocytes. This, when using a combination of the present marker(s) and the CD8beta marker, CD4 and CD8 cells can be specifically distinguished. This was not possible before the invention, since all CD4 looked identical to non-T lymphocytes.
The invention will now be further described based on the following examples and with reference to the accompanying figures and the sequence protocol, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures and Sequences,

EXAMPLES

Example 1—CD3-Analysis

The inventors have purified various blood subsets, including CD3/CD4, CD3/CD8 naïve and memory T lymphocytes, CD56 natural killer cells, CD19 naïve and memory B cells, CD14 monocytes and CD15 granulocytes. DNA from the purified cells was bisulfite-treated and analysed at various CpG dinucleotide motifs. The inventors then compared the methylation status (finding C as for Cytosine that was methylated in the original sequence versus T for cytosine that was unmethylated in the original sequence).
The data showed various CpG motifs and areas in the CD3 γ, δ and ε that were demethylated in all CD3CD4 and CD3CD8 cell types while methylated in all other blood cell types. The differentially methylated gene regions as found for CD3 γ, δ and ε are shown below in FIG. 1 and are indicated in bold as “blast hits.”
The data, as observed with Illumina Golden Gate technology, show that all CD4 and CD8 positive memory (0.06 and 0.06 respectively) and naïve (0.03 and 0.06, respectively) T cells are subject to much lower methylation rates than all other tested cell types, including CD15 (0.92), CD14 (0.90), CD19 memory and naïve (0.81 and 0.67, respectively), CD56 (0.86) positive blood cells as well as cells derived (0.78) from non-blood tissues. The experimental results are further depicted in the following table.

TABLE 1

Methylation analysis results

		S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	S11	S12
		n = 12;	n = 5;	Pool;	Pool;	Pool;	Pool;	Pool;	Pool;	Pool;	Pool;	Pool;	Pool;
Gene	Chr.	female	male	female	(5 male)	(5 male)	(5 male)	(5 male)	(5 male)	(5 male)	(5 male)	(5 male)	(5 male)

CD3D	11	0.8	0.6	0.6	0.9	0.9	0.9	0.0	0.1	0.1	0.1	0.7	0.8
		0.9	0.7	0.7	0.9	0.9	0.9	0.1	0.1	0.2	0.2	0.7	0.8
CD3G	11	0.7	0.6	0.7	0.9	0.9	0.8	0.1	0.1	0.1	0.1	0.8	0.9
		0.4	0.6	0.5	0.7	0.8	0.7	0.1	0.1	0.1	0.1	0.3	0.6

S1 = ovarian tissue,
S2 = whole blood,
S3 peripheral blood mononuclear cells,
S4 = granulocytes,
S5 = monocytes,
S6 = NK cells,
S7 = naïve T helper cells,
S8 = memory T helper cells,
S9 = naïve cytotoxic T cells,
S10 = memory cytotoxic T cells,
S11 = naïve B-cells,
S12 = memory B-cells.,
Chr = chromosome

The data showing the high specificity were obtained using a Illumina Golden Gate technology, with the following genomic CpGs regions analysed

CD3γ

cg15880738

(SEQ ID NO: 2)

(+1)AGCTGCTGCACAGGCTGGCTGGCTGGCTGGCTGCTAAGGGCTGCTC

CA CG

cg07545925

(SEQ ID NO: 3)

(+1) CG GAAAAACAAAAGGCATCTGCACCTGCAGCCCTGCTGAGGCCCCT

GCTG

CD3δ

cg24841244

(SEQ ID NO: 4)

(−1) ACCCAGGCTGATAGTT CG GTGACCTGGCTTTATCTACTGGATGAG

TTC CG

cg07728874

(SEQ ID NO: 5)

(−1) TGGAACATAGCA CG TTTCTCTCTGGCCTGGTACTGGCTACCCTTC

TCT CG

CD3ε

cg24612198

(SEQ ID NO: 9)

AGTCATCTGTTTTGCTTTTTTTCCAGAAGTAGTAAGTCTGCTGGCCTCCG

Since the Illumina technology does only allow the analysis of a single CpG (or rather 2 CpGs per gene locus) the inventors verified the methylation properties using bisulfite sequencing. For that, the inventors bisulfite treated the samples using the Qiagen EpiTect kit, and sequenced the samples using an ABI 3100 prism sequencer. For data interpretation, the KB base calling software supplied by ABI was used.
While not providing entirely quantitative results calculating the percentile of methylated (i.e., CG signal on the plus strand sequence) versus unmethylated (i.e., TG on the plus strand sequence), the data were unambiguous for the purified cell types. All T-lymphocytes were overwhelmingly demethylated at all CG positions analysed, whereas all other analysed cell types were methylated at identical positions.

Example 2—Analysis of Additional Markers in Analogy to CD3

In order to identify further suitable markers distinguishing and monitoring T-lymphocytes, other markers in addition to CD3 have been identified and tested through methylation analysis. It was found that methylation in the CpG positions in the genes for SLA2, CHRNA3, C16orf24, LCK, FASLG, CD7, SIT1, IL32, CXCR6, UBASH3A, GRAP2, ITGB7 and TXK can also be used in the context of the present invention, as these markers are also able to identify CD3 positive T lymphocytes.
Furthermore, other markers have been identified that identify the subset of the CD8 and CD4 positive cells in the group of CD3 positive T lymphocytes. The genes for GNGT2, CRTAM, IL2RB and ZBTB32 have been found to segregate between CD8 and CD4 positive cells. Equivalently, FLJ00060, FLJ38379, PPP6C, CD226, ZBTB7B and TNFAIP8 are capable of positively identifying CD4 expressing cells in whole blood and segregate between CD4 and CD8 positive CD3 positive cells.
The following Table 2 summarizes the Illumina-data as obtained for the above markers at selected CpG positions. It can be seen that several other markers can be used in order to selectively identify CD4+(for CD3+ lymphocyte subset-identification), namely FLJ00060; FLJ38379; PPP6C; CD226; ZBTB7B and/or TNFAIP8, in order to selectively identify CD8+(for CD3+ lymphocyte subset-identification), namely GNGT2; CRTAM; IL2RB and/or ZBTB32, and in order to selectively identify CD3+ lymphocytes, namely CD3D; CD3G, and/or CD3E, and/or SLA2, CHRNA3, C16orf24; LCK; FASLG; FASLG; CD7; SIT1; IL32; CXCR6; UBASH3A; GRAP2; ITGB7 and/or TXK, as shown with selected CpG sites as preferred examples. Based on the table, the person of skill will be able to extend the teaching regarding CD3 as herein to these markers and their CpG sites.

TABLE 2

Illumina-data as obtained selected markers at CpG positions Chr. = chromosome

			SEQ ID	Ovar
			No./	Tissue	Whole		BCST18	BCST19	BCST20
	Gene		Accession	(mean,	Blood	PBMC	Granulocyte	Monocyte	NK
	Name	Chr.	No	n = 12)	(Pool)	(Promega)	(CD15+)	(CD14+)	(CD56+)

CD4+
marker
CpG-ID
cg03602500	FLJ00060	19	10/	0.802	0.728	0.779	0.858	0.851	0.873
			NM_033206.1
cg16173109	FLJ38379	2	11/	0.614	0.675	0.719	0.833	0.844	0.784
			XR_001026.1
cg00620024	PPP6C	9	12/	0.641	0.619	0.692	0.801	0.851	0.836
			NM_002721.3
cg13164537	CD226	18	13/	0.458	0.470	0.508	0.582	0.557	0.601
			NM_006566.1
cg01782486	ZBTB7B	1	14/	0.789	0.725	0.720	0.823	0.809	0.886
			NM_015872.1
cg07086380	TNFAIP8	5	15/	0.728	0.579	0.635	0.857	0.733	0.793
			NM_014350.1
CD8+
marker
CpG-ID
cg17839611	GNGT2	17	16/	0.779	0.629	0.688	0.828	0.867	0.207
			NM_031498.1
cg22512531	CRTAM	11	17/	0.651	0.634	0.709	0.835	0.735	0.5
			NM_019604.2
cg26757673	IL2RB	22	18/	0.799	0.662	0.752	0.878	0.9	0.092
			NM_000878.2
cg08539991	ZBTB32	19	19/	0.823	0.736	0.791	0.912	0.926	0.674
			NM_014383.1
CD4+/
CD8+
marker
CpG-ID
cg24841244	CD3D	11	20/	0.783	0.582	0.644	0.917	0.904	0.857
			NM_000732.3
cg07728874	CD3D	11	21/	0.854	0.683	0.727	0.911	0.901	0.881
			NM_000732.3
cg15880738	CD3G	11	22/	0.714	0.616	0.682	0.874	0.876	0.836
			NM_000073.1
cg07545925	CD3G	11	23/	0.370	0.552	0.545	0.737	0.771	0.739
			NM_000073.1
cg24612198	CD3E	11	24/	0.679	0.485	0.563	0.794	0.793	0.698
			NM_000733.2
cg04759756	SLA2	20	25/	0.857	0.673	0.754	0.892	0.925	0.711
			NM_032214.2
cg22670733	CHRNA3	15	26/	0.792	0.733	0.721	0.911	0.888	0.8
			NM_000743.2
cg09830866	C16orf24	16	27/	0.493	0.629	0.647	0.842	0.79	0.076
			NM_023933.1
cg17078393	LCK	1	28/	0.807	0.575	0.666	0.920	0.884	0.26
			NM_005356.2
cg10161121	FASLG	1	29/	0.704	0.587	0.697	0.905	0.892	0.068
			NM_000639.1
cg00071250	FASLG	1	30/	0.659	0.498	0.602	0.871	0.838	0.075
			NM_000639.1
cg02473123	CD7	17	31/	0.811	0.681	0.767	0.942	0.898	0.339
			NM_006137.6
cg15518883	SIT1	9	32/	0.817	0.601	0.696	0.911	0.885	0.9033
			NM_014450.2
cg18350391	IL32	16	33/	0.332	0.724	0.794	0.937	0.901	0.658
			NM_001012631.1
cg25226014	CXCR6	3	34/	0.702	0.481	0.548	0.789	0.89	0.35
			NM_006564.1
cg13578652	UBASH3A	21	35/	0.564	0.444	0.475	0.537	0.703	0.169
			NM_018961.2
cg25712380	GRAP2	22	36/	0.633	0.602	0.639	0.824	0.83	0.506
			NM_004810.2
cg19812619	ITGB7	12	37/	0.819	0.651	0.785	0.874	0.897	0.645
			NM_000889.1
cg02600394	TXK	4	38/	0.775	0.576	0.682	0.867	0.909	0.082
			NM_003328.1

		BCST22	BCST23	BCST24			Mean
	BCST21	T mem	CTL	CTL			Value		Delta
	T naive	(CD4+	naive	mem	BCST25	BCST26	Target	Mean	Meth
	(CD4+ CD27+	CD27+	(CD8+ CD27+	(CD8+ CD27+	B naive	B mem	Cell	Value	(Target-
	CD45RA+)	CD45RA−)	CD45RA+)	CD45RA−)	(CD19+)	(CD19+)	Type	Rest	Rest)

CD4+
marker
CpG-ID
cg03602500	0.235	0.279	0.521	0.687	0.656	0.747	0.257	0.75	−0.493
cg16173109	0.1141	0.113	0.341	0.383	0.418	0.385	0.113	0.599	−0.486
cg00620024	0.107	0.169	0.269	0.420	0.404	0.502	0.138	0.603	−0.465
cg13164537	0.029	0.071	0.351	0.543	0.366	0.501	0.05	0.494	−0.443
cg01782486	0.190	0.428	0.875	0.864	0.586	0.414	0.309	0.749	−0.44
cg07086380	0.110	0.106	0.288	0.148	0.309	0.208	0.108	0.528	−0.419
CD8+
marker
CpG-ID
cg17839611	0.340	0.562	0.09	0.119	0.854	0.722	0.104	0.648	−0.543
cg22512531	0.363	0.630	0.079	0.082	0.194	0.336	0.08	0.559	−0.478
cg26757673	0.573	0.086	0.157	0.061	0.411	0.403	0.109	0.555	−0.446
cg08539991	0.563	0.153	0.303	0.085	0.648	0.140	0.194	0.637	−0.442
CD4+/
CD8+
marker
CpG-ID
cg24841244	0.025	0.057	0.057	0.058	0.673	0.810	0.049	0.771	−0.721
cg07728874	0.128	0.149	0.175	0.155	0.744	0.807	0.152	0.814	−0.661
cg15880738	0.061	0.091	0.093	0.103	0.811	0.898	0.087	0.788	−0.701
cg07545925	0.133	0.149	0.132	0.131	0.303	0.601	0.132	0.49	−0.358
cg24612198	0.064	0.036	0.093	0.051	0.206	0.279	0.061	0.562	−0.501
cg04759756	0.363	0.228	0.129	0.211	0.756	0.849	0.233	0.802	−0.569
cg22670733	0.122	0.180	0.092	0.315	0.456	0.668	0.177	0.746	−0.568
cg09830866	0.041	0.056	0.033	0.058	0.72	0.510	0.047	0.588	−0.541
cg17078393	0.05	0.037	0.036	0.041	0.144	0.27	0.041	0.566	−0.524
cg10161121	0.051	0.06	0.088	0.06	0.353	0.441	0.065	0.581	−0.516
cg00071250	0.061	0.069	0.139	0.051	0.391	0.41446208	0.08	0.543	−0.463
cg02473123	0.102	0.328	0.168	0.372	0.767	0.81	0.243	0.752	−0.509
cg15518883	0.11	0.142	0.154	0.358	0.392	0.373	0.191	0.697	−0.506
cg18350391	0.296	0.169	0.15	0.192	0.881	0.406	0.202	0.704	−0.502
cg25226014	0.028	0.046	0.046	0.193	0.258	0.514	0.078	0.567	−0.488
cg13578652	0.027	0.04	0.035	0.05	0.602	0.673	0.038	0.521	−0.483
cg25712380	0.097	0.093	0.089	0.1	0.179	0.310	0.095	0.565	−0.470
cg19812619	0.3374	0.275	0.199	0.177	0.608	0.4337	0.247	0.714	−0.467
cg02600394	0.036	0.075	0.051	0.354	0.376	0.484	0.129	0.594	−0.465

Claims

We claim:

1. A method for identifying CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes in a sample derived from a human comprising T cells, the method comprising:

a) obtaining a sample comprising T cells from a human,

b) isolating DNA from said T cells comprising a sequence according to SEQ ID NO: 1, and treating said DNA with bisulfite,

c) amplifying an amplicon from said treated DNA using bisulfite-specific primer pairs according to SEQ ID NOs: 70 and 71 and SEQ NOs: 72 and 73,

d) analyzing the methylation status of at least one CpG position in said amplicon as amplified in step c), and

e) identifying CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocyte cell.

2. The method according to claim 1, wherein said at least one CpG position is demethylated to more than 91%.

3. The method according to claim 1, further comprising the step of discriminating said CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes from other leukocytes in said sample based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocyte cell.

4. The method according to claim 1, wherein said analysis of the methylation status further comprises a method selected from bisulfite sequencing, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE.

5. The method according to claim 1, wherein said method is suitable for application on a DNA-chip.

6. The method according to claim 1, wherein said identification comprises a distinction of said T-lymphocytes from all major peripheral blood cell types or non-blood cells.

7. The method according to claim 1, wherein said human suffers from or is likely to suffer from an autoimmune disease, a transplant rejection, cancer, and/or an allergy.

8. The method according to claim 1, the method further comprising the steps of:

f) quantifying the amount of CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes in said sample from step a) based on said identification in step e),

g) quantifying the amount of T-lymphocytes in an earlier sample from said human or in a control sample comprising an identification according to steps b) to e), and

h) comparing the amount of T-lymphocytes in the present sample from step (a) to said earlier sample or to the control sample.

9. The method according to claim 8, wherein said human suffers from or is likely to suffer from an autoimmune disease, a transplant rejection, cancer, and/or an allergy.

10. The method according to claim 1, wherein said method further comprises analysing the amplicon to determine if it has sequences according to SEQ ID NOs: 6, 7, or 8.

11. A method for quantifying the methylation of at least one CpG position in a sequence according to SEQ ID NO: 1, the method comprising:

a) obtaining a sample comprising T cells from a human,

c) amplifying an amplicon from said treated DNA using bisulfite-specific primer pairs according to SEQ ID NOs: 70 and 71 and SEQ ID NOs: 72 and 73,

d) quantifying the methylation of the at least one CpG position in said amplicon as amplified in step c),

wherein a demethylation of the least one CpG position in said amplicon to at least 90% is indicative for a CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocyte cell.

12. The method, according to claim 11, wherein step d) comprises quantifying the methylation of a combination of at least two CpG positions in said amplicon as amplified in step c),

wherein a demethylation of a combination of at least two CpG positions in said amplicon to at least 90% is indicative for a CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocyte cell.

13. A method for identifying CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes in a sample derived from a human comprising T cells, the method comprising:

a) obtaining a sample comprising T cells from a human,

c) performing quantitative Polymerase Chain Reaction on the bisulfite-treated DNA using bisulfite-specific primer pairs and a bisulfite-specific probe,

e) identifying CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ naïve and/or memory T-lymphocytes based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3⁺ CD4⁺ and/or CD3⁺ CD8⁺ nave and/or memory T-lymphocyte cell.

14. The method, according to claim 13, wherein the bisulfite-specific primer pair is SEQ ID NOs: 76 and 77 and the bisulfite-specific probe is SEQ ID NO: 78.

15. The method, according to claim 13, wherein the bisulfite-specific primer pair is SEQ ID NO: 79 and 80 and the bisulfite-specific probe is SEQ ID NO: 81.

16. An oligomer having to any of SEQ ID NOs: 70 to 73, 76, 77, 79, and 80 or an amplicon having SEQ ID NO: 6 to 8.

17. A kit for identifying and/or monitoring CD3⁺ T-lymphocytes, in particular CD3⁺ CD4⁺, or CD3⁺ CD8⁺ T-lymphocytes, in a mammal based on the analysis of the methylation status of CpG positions in the gene CD3, comprising materials for performing a method according to claim 1.

18. The kit according to claim 17, comprising a) a bisulfite reagent, and b) materials for the methylation analysis of CpG positions selected from the positions consisting of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of an amplicon according to SEQ ID NO: 6, 7, or 8.