Patent Application
20070160994
The present invention provides a process for determining the quantitative and qualitative profile of the repertoire of a given type of an immunoglobulin (Ig) heavy chain expressed by a lymphocyte B population present in a tissue sample, and kits and uses thereof. It also provides a set of VH forward primers associated with a CH reverse primer, which are respectively capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the variable segments of Ig heavy chains and with the constant segment of a given type of Ig heavy chain, such as preferably IgM, IgG, IgE or IgA heavy chain. Methods for the in vitro diagnosis of a condition associated with an abnormal expression of the repertoire of a given type of Ig heavy chain, and for the in vitro follow-up of a treatment of such a condition, are also comprised herein.
An essential feature of the immune system is the capacity to recognize specifically a large number of antigens. In vertebrates, the B lymphocytes partly execute this function of recognition, by means of the immunoglobulins.
To the tremendous variety of antigens to be recognized, there corresponds a very wide potential diversity of these immunoglobulins. Indeed, immunoglobulins are composed of four peptide chains, the NH2 terminal domains of which are highly variable. It is the existence of a strong interaction between a given antigenic determinant and the site consisting of these variable domains which constitutes the expression at molecular level of the phenomenon of recognition. Thus, the information contained in the genome of a mouse enables it to produce potentially at least 1011 immunoglobulins of different variable region.
The notion of repertoire thus emerges: the set of immunoglobulin variable regions present at a given instant in an organism for a given type of immunoglobulin constitutes the current repertoire of immunoglobulins of said given type.
The diversity of B cell repertoire is strictly correlated with the diversity of the antibodies they express and produce. Antibody diversity is achieved in multiple ways. The rearrangement of V gene segments together with D and J segments for the heavy chain and V genes and J segments for the light chain allows combinational diversity by association of different sets of genes. The two recombinase activating genes RAG1 and RAG2 are responsible for the V(D)J recombination process (Schatz and al. (1989). Cell 59, 1035-48; Oettinger and al. (1990). Science 248, 1517-23). Imprecise junctions of those gene segments, either by nibbling or by random addition of nucleotides by the Tdt enzyme increase again the diversity level (Bollum, F. J. (1978). Adv Enzymol Relat Areas Mol Biol 47, 347-74; Komori and al. (1993). Science 261, 1171-5; Gilfillan and al. (1993). Science 261, 1175-8). Antigen-driven affinity maturation introducing somatic hypermutations (SHM) in the V region is another step in generating diversity, as well as the process of heavy and light chain pairing (Wu and al. (2003). J Clin Immunol 23, 235-46). Class switch recombination (CSR), resulting in the production of several antibody isotypes, increases the diversity and functionality of the B cell repertoire, allowing one given variable region to be associated with different constant regions (Honjo and al. (2002) Annu Rev Immunol 20, 165-96). Finally, in some species, antibody diversification is mainly achieved by gene conversion (Thompson and Neiman (1987). Cell 48, 369-78; Reynaud and al. (1987). Cell 48, 379-88). Recently, it has been demonstrated that the three processes, SHM, CSR and gene conversion, require a single enzyme, the activation-induced cytidin desaminase (AID) (Muramatsu and al. (2000). Cell 102, 553-63; Revy and al. (2000). Cell 102, 565-75; Arakawa and al. (2002). Science 295, 1301-6; Okazaki and al. (2002). Nature 416, 340-5; Yoshikawa and al. (2002). Science 296, 2033-6). While V(D)J recombination and Tdt activity contribute to diversity generation of both T and B cell repertoires (Tuaillon and Capra (2000). J Immunol 164, 6387-97; Cabaniols and al. (2001). J Exp Med 194, 1385-90), SHM, CSR and gene conversion are B cell specific mechanisms. These additional mechanisms could eventually account for an increased diversity of the B cell repertoire as compared with that of T cells.
It represents a monumental task to describe the collective antibodies (Ab or immunoglobulins Ig) expressed at a given moment in a subject, since the immunoglobulin repertoire probably contains millions of different molecules. Only a small number of reagents capable of specifically recognizing elements of such a repertoire is as yet available. It is, of course, possible to work more finely by determining the sequence of a certain number of expressed genes. However, practical considerations make it scarcely conceivable to analyze routinely more than about ten or, perhaps, a hundred genes, and the operation is expensive and very lengthy. In short, the repertoire of immunoglobulins is described at the present time only by means of a small number of parameters.
Hence methods are not available which permit a rapid and effective analysis of the physiological and pathological situations associated with the statement of these repertoires. For example, it is clear that these repertoires vary during a voluntary immunization (vaccine), or during infection by pathogenic microorganisms, or during the progress of autoimmune pathologies. In the latter case, there are many reasons to believe that predisposition to these diseases mirrors a certain composition of the repertoires. It is hence very probable that a good method of analysis of the repertoires would have medical spin-offs, and could form the basis of techniques of medical analysis and of diagnosis.
Human T cell repertoire diversity has been analysed using different approaches (see the european patents granted under the numbers EP 566685 and EP 672184). Limiting dilutions analyses have shown that CD4 T cells from healthy individuals display a highly diverse TCR-β repertoire (Wagner and al. (1998). Proc Natl Acad Sci U S A 95, 14447-52). Based on Immunoscope technologies, the inventors have estimated the lower limit of the total αβ TCR diversity in humans to be 25×106 different TCRs (Arstila and al. (1999). Science 286, 958-61]. Furthermore, the overall diversity of TCR-β memory CD8 T cell repertoire was estimated to be 5.104 to 105 clonotypes (Arstila and al. (1999). Science 286, 958-61; Baron and al. (2003). Immunity 18, 193-204). On the contrary, little is known about the size and diversity of the peripheral blood human B cell repertoire. Most of the studies on B cell repertoire have been performed either in pathological situations or following repeated immunizations, preventing any extrapolation on the global size and diversity under physiological conditions.
The contribution of somatic mutations to B cell repertoire diversity has been widely studied, but most studies have been carried out in murine models, determining the proportion of a mutated VH gene by a comparison with its germline sequence, as first shown by Gojobori et al. (1986, Mol Biol Evol 3, 156-67). The conclusions of those studies was that peripheral blood lymphocytes from most mice are umnutated (less than 5%) (Schittek and Rajewsky (1992). J Exp Med 176, 427-38) and that most mutated B lymphocytes are localized in germinal centers (Berek and al. (1991). Cell 67, 1121-9; MacLennan and Gray (1986). Immunol Rev 91, 61-85). A more recent study has focused on the contribution of somatic mutations to the diversity of murine serum immunoglobulins (Williams and al. (2000). Immunity 13, 409-17). Mutations do not seem to be involved in the diversity of serum IgM, IgG and IgA in young mice, but they accumulate with age in response to environmental antigens and in much higher proportions within IgG than in IgM. (Williams and al. (2000). Immunity 13, 409-17). In humans, the proportion of peripheral blood B lymphocytes bearing somatic mutations is much higher compared to mice, reaching up to 40% of all peripheral B lymphocytes, and they were shown to express the CD27 marker. (Klein and al. (1997). Blood 89, 1288-98; Klein and al. (1998). J Exp Med 188, 1679-89). However, these studies did not bring information on the global B cell clone size and on the proportion of clonal expansion within a non antigen selected rearrangement.
Similarly, two studies have been conducted for the immunoglobulins, namely Loembé and al, Eur J Immunol. December 2002;32(12):3678-88, and Fais and al, J Clin Invest. Oct. 15, 1998;102(8):1515-25.
The immunoscope method, based on PCR techniques, allows the quantitative study of the lymphocytes T repertoires by determining the length of the CDR3 regions and TCR, quantifying the use of the variable segments and optionally elucidating their complete sequences. The use of these methodologies for studying the human immunoglobulin repertoires and the possibility to quantify the use of the variable segments in the formation of the immunoglobulin heavy chains, although often mentioned, have never realized.
The object of the present invention is thus to provide the tools which are necessary for enabling the quantitative study of the lymphocyte B repertoire in mammals. These studies can be conducted in physiological conditions, for example within the context of the follow-up of a vaccine whose protective power comes from the production of specific antibodies, or in pathological conditions, for example when an auto-immune disease arises with the production of autoantibodies. Thus, the new application range described herein allows the follow-up of pathologies which are specifically derived from the immunoglobulin expression by the B lymphocytes and therefore distinct from that which are specifically derived from the T lymphocyte expression.
Thanks to their sensibility, the process of the present invention allows to determine the quantitative and qualitative profile of the repertory of a given type of an immunoglobulin heavy chain expressed by a lymphocyte B population, thereby determining the specific clones of lymphocyte B and the nucleic acid sequences of the genes encoding the immunoglobulins. This determination allows to follow more individually each of the cellular clones in various samplings of a given subject.
The present invention provides advantages over the prior art: firstly, the obtention of quantitative results doesn't require DNA sequencing, unlike the “unique cell PCR technique”, which is much more difficult to perform and which is only available with populations previously sorted. Secondly, the results can be deduced from a large number of cells and thus are more significative in a statistical point of vue, always in comparison with the “unique cell PCR technique”. Thirdly, because no antibody specific for the VH subgroups exists, the FACS quantification technique used for studying the specific lymphocyte T Vβ subgroups is not applicable to the B lymphocytes. And finally, unlike the ELISA technique which allows to describe at the proteic level the specificity and the type of the circulating antibodies, the B immunoscope technology allows to quantitatively analyse at the mRNA level the repertoire of the intracytoplasmic immunoglobulins or of the immunoglobulins expressed at the surface of the B lymphocytes, thereby providing earlier and more detailed informations of the activity state of the genes concerned.
As a consequence, the present invention provides the obtention of quantitative and qualitative informations directly from the lymphocyte B repertoire without any sequencing, it provides a rapid method, and gives the possibility to distinguish and quantify the VH repertoire specific of each immunoglobulin type individually or as a whole.
Thus, a first object of the present invention is to provide a process for determining the quantitative and qualitative profile of the repertoire of a given type of an immunoglobulin heavy chain expressed by a lymphocyte B population present in a tissue sample, characterized in that it comprises the following steps:
The terms of antibody and immunoglobulin, which are indifferently used herein, refer to the association of two heavy chains and two light chains. The part of the antibody which is specific for an antigen is constituted by the rearrangement of three gene segments V D and J for the heavy chains and V and J for the light chains. The variable segments VH of the heavy chains are classified in VH subgroups relative to the sequences of the nucleic acids which encode them. In humans, the VH subgroups are at least the VH1, VH2, VH3a, VH3b, VH4, VH5, VH6 and VH7 subgroups. Accordingly, the JH subgroups are at least the JH1, JH2, JH3, JH4, JH5 and JH6 subgroups.
The quantitative and qualitative profile of the repertoire of a given type of an immunoglobulin heavy chain refers in the present invention to the profile corresponding at once to the relative use of the nucleic acids encoding the heavy chains of immunoglobulins of a given type, in particular the nucleic acids encoding the variable segments (VH) of immunoglobulin heavy chains of a given type, expressed by a lymphocyte B population and to the length of the VDJ rearrangements for each VH or JH subgroup.
The repertoire of a given type of an immunoglobulin heavy chain refers to the immunoglobulin heavy chains of a given type which are expressed by a lymphocyte B population present in a tissue sample.
The type, or class, of an immunoglobulin heavy chain may be any type which is sufficiently described in the literature, such as the IgM type, the IgG type, in particular the IgG1 type, the IgG2 type, the IgG3 type, or the IgG4 type, the IgE type, the IgA type or the IgD type. Thus, for example, the quantitative and qualitative profile will be determined for the IgM type with the process according to the present invention.
Indeed, the lymphocytes B are subjected to the isotypic commutation: for the same variable segment, an immunoglobulin can belong to a given type and so have different properties relative to its type. The IgM are the most present immunoglobulins in the serum, the IgG correspond generally to an anamnestic reaction, the IgA are mostly expressed by the gut mucosa and the IgE are characteristic for an allergic response.
The size of a B lymphocyte clone may vary in an important manner in the course of time or after an immune response. The process for determining the quantitative and qualitative profile of the present invention allows to detect directly, without inevitably sequencing them, the presence of clonal expansions in a given rearrangement.
The tissue sample comprising the lymphocyte B population is usually the lymphocyte B population present in the blood of a subject, but it be may also tissue sample of the lymphoid system, such as for example lymph nodes or tonsil. The process for determining the quantitative and qualitative profile of the present invention doesn't necessarily require to previously purify the lymphocyte B population from the tissue sample, thus allowing to greatly facilitate its implementation. Accordingly, the process may be performed from a heterogeneous cellular population, but also from a previously purified lymphocyte B population, or from a lymphocyte B subpopulation, such as for example naive B lymphocytes versus the memory B cells or according to the antigenic specificity antibodies they express, and therefore performing previous classical purification techniques such as magnetic sorting or any cellular sorting technique.
The cellular nucleic acid content of the tissue sample is then obtained from the tissue sample using conventional techniques. When this nucleic acid content is mRNA, the cDNA synthesis performed thanks to the reverse transcription reaction is well known, as well as the method allowing to only transcribe the mRNA present in the total RNA of the tissue sample thanks to a poly(T)primer (see Example 1: “RNA and cDNA preparation”). The nucleic acid content may also be the DNA extract of the tissue sample (see EP 566685).
Methods of DNA engineering are sufficiently described in the literature, and are well known by the man skilled in the art, who will know how to use them in the most convenient manner in the process of the invention (see Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; by Ausubel et al. (1994), eds Current Protocols in Molecular Biology, Current Protocols Press; and by Berger and Kimmel (1987), Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, Calif. the disclosures of which are hereby incorporated by reference).
Then the amplification is performed using a PCR type technique, that is to say the PCR technique or any other apparented technique: two primers (VH and CH), respectively complementary to the variable segment and to the constant segment of a given type of Ig heavy chain are then added to the nucleic acid content (cDNA or DNA) along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the VH and CH primers.
The expression “specifically hybridizing in stringent conditions” refers in the present invention to a hybridizing step in the process of the invention where the oligonucleotide sequences selected as probes or primers are of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur during the amplification.
Hybridization is typically accomplished by annealing the oligonucleotide probe or primer to the cDNA (or DNA) under conditions of stringency that prevent non-specific binding but permit binding of this cDNA which has a significant level of homology with the probe or primer.
Among the conditions of stringency is the melting temperature Tm for the amplification step using either the set of VH forward primers associated with the CH primer, or the VH internal forward primer associated with the set of JH reverse primers, which is in the range of about 58° C. to about 60° C.; preferably, the Tm for the amplification step is in the range of about 58° C. or about 60° C. Most preferably, the Tm for the amplification step is about 60° C.
Typical hybridization and washing stringency conditions depend in part on the size (i.e., number of nucleotides in length) of the cDNA or oligonucleotide probe (Ausubel and al., 1994, eds Current Protocols in Molecular Biology).
The oligonucleotide probes or primers herein described may be prepared by any suitable methods such as chemical synthesis methods (see references supra for methods of DNA engineering).
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that separated amplifications are performed for each of the VH subgroups.
Indeed, the nucleic acid content (cDNA or DNA) obtained at the step (a) from the tissue sample is divided such as to perform so much separated amplifications as there are couples of VH and CH primers.
By the expression mixture of CH reverse primer it is intended to refer in the present invention to a mixture of at least two, preferably at least 2, 3, 4 or at least 5 CH reverse primers, said CH reverse primers being preferably present in an equivalent quantity between each other. For example, when there are 2 CH reverse primers in the mixture, each CH reverse primer is present at 50% in said mixture.
In a preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the separated amplifications are real-time separated amplifications, said real-time amplifications being performed using a CH labeled reverse probe, preferably a CH labeled reverse hydrolysis-probe, capable of specifically hybridizing in stringent conditions with the constant segment of the given type of immunoglobulin heavy chain and capable of emitting a detectable signal everytime each amplification cycle occurs, and characterized in that the signal obtained for each VH subgroup is measured.
The real-time amplification, such as real-time PCR, is well known by the man skilled in the art, and the various known techniques will be employed in the best way for the implementation of the present process. These techniques are performed using various categories of probes, such as hydrolysis probes (TaqMan, Applied Biosystems, USA), hybridization adjacent probes, or molecular beacons. Hydrolysis probes are preferred. The techniques employing hydrolysis probes or molecular beacons are based on the use of a fluorescence quencher/reporter system, and the hybridization adjacent probes are based on the use of fluorescence acceptor/donor molecules.
Hydrolysis probes with a fluorescence quencher/reporter system are available in the market, and are for example commercialized by the Applied Biosystems group (USA). Many fluorescent dyes may be employed, such as FAM dyes (6-carboxy-fluorescein), or any other dye phosphoramidite reagents. In the present invention, the inventors have employed a TaqMan Minor Groove Binder (MGB) FAM-labeled probe (see Example 1), but other probes may be used, in a convenient manner.
Among the stringent conditions applied for anyone of the hydrolysis-probes of the present invention is the Tm, which is in the range of about 68° C. to 70° C.; preferably, the Tm for anyone of the hydrolysis-probes of the present invention is in the range of about 69° C. to about 70° C. Most preferably, the Tm applied for anyone of the hydrolysis-probes of the present invention is about 70° C.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the separated amplification products obtained for each of the VH subgroups are further elongated using a CH labeled reverse probe capable of specifically hybridizing in stringent conditions with the constant segment of the given type of immunoglobulin heavy chain and capable of emitting a detectable signal, and characterized in that the elongation products are separated, for each of the VH subgroups, relative to their length, the signal obtained for the separated elongation products is measured, and the quantitative and qualitative profile of the labeling intensity relative to the elongation product length is established, for each of the VH subgroups individually.
This elongation step, also named “run-off reaction”, allows to determine the length of the VDJ rearrangements from each VH subgroup. The separated amplification products obtained for each VH subgroup are elongated using a DNA polymerase and a CH labeled reverse probe capable of specifically hybridizing in stringent conditions with the constant segment of the given type of immunoglobulin heavy chain and capable of emitting a detectable signal. Elongation products are thus obtained, which are labeled at their CH end and their length can be determined.
The determination of the length can be realized using conventional techniques for the determination of DNA sequences, with gels such as polyacrylamide gels for the separation, DNA sequencers and adapted softwares. The labeling of the CH labeled reverse probe may be any appropriate labeling, such as for example radio-labeling or preferably fluorescent-labeling. The “run-off reaction” is well described in EP 566 685.
Among the stringent conditions applied for anyone of the labeled probes used for the elongation (“run-off reaction) is the Tm, which is in the range of about 58° C. to about 60° C. ; preferably, the Tm for the amplification step is in the range of about 58° C. or about 60° C. Most preferably, the Tm for the amplification step is about 60° C.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the set of VH forward primers comprises at least the 8 following subgroups of VH primers corresponding to the VH subgroups:
More preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the sequences SEQ ID No 1 to SEQ ID No 15 may contain at least one to three point mutations, except for the nucleotides 1 to 6 of their 3′ part.
The point mutation refers in the present invention to a mutation which occurs for only one nucleotide, on the contrary of a mutations which occurs for an oligonucleotide sequence. The point mutation may be a substitution, a deletion or an addition.
The process for determining the quantitative and qualitative profile according to the present invention may further be characterized in that the CH reverse primer is selected from the CH reverse primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the constant segments (CH) of the IgM heavy chain, the IgE heavy chain, the IgG heavy chain and the IgA heavy chain.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgM heavy chain, the CH reverse primer has the sequence SEQ ID No 26, or the sequence SEQ ID No 26 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgE heavy chain, the CH reverse primer has the sequence SEQ ID No 33, (HIGCGE1), or the sequence SEQ ID No 33 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgE heavy chain, the CH reverse primer has the sequence SEQ ID No 42, (HIGCGE4), or the sequence SEQ ID No 42 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is the IgG type, a mixture of two CH reverse primers is associated with the set of VH forward primers, said two CH reverse primers having the sequences SEQ ID No 27 and SEQ ID No 28, or the sequences SEQ ID No 27 and SEQ ID No 28 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention may further be characterized in that the CH reverse primer is selected from the CH reverse primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the constant segments (CH) of all IgG (IgG1, IgG2, IgG3 and IgG4).
Indeed, the CH reverse primers HIGCG int 1 (SEQ ID No40) and HIGCG int 2 (SEQ ID No41) are preferably used to analyse IgG (1 to 4) subclasses. Evenmore, HIGCG int 2 (SEQ ID No41) is better than HIGCG int 1 (SEQ ID No40) to analyse the quantitative and qualitative profile of IgG subclasses. It has to be noted that HIGCG int 1 may be used to sequence VH and CDR3 segments.
Thus, the process for determining the quantitative and qualitative profile of the present invention may be characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgG heavy chain, the sequence of the CH reverse primer is selected from the group consisting of SEQ ID No40 and SEQ ID No41, or SEQ ID No 40 and SEQ ID No41 wherein one to tree point mutations may occur, except for the nucleotides 1 to 6 of their 3′ part. In a most preferred embodiment, the CH reverse primer is SEQ ID No41.
Furthermore, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgM heavy chain and when the separated amplifications are real-time separated amplifications, the CH labeled hydrolysis-probe has the sequence SEQ ID No 29, or the sequence SEQ ID No 29 wherein at least one point mutation may occur.
Furthermore, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgM heavy chain and when the separated amplification products obtained for each of the VH subgroups are further elongated, the CH labeled reverse probe has the sequence SEQ ID No 30, or the sequence SEQ ID No 30 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the CH labeled reverse probe SEQ ID No30 is fluorenscent on its 5′ part.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgE heavy chain, when the CH reverse primer has the sequence SEQ ID No33 (HIGCGE1), and when the separated amplifications are real-time separated amplifications, the CH labeled hydrolysis-probe has the sequence SEQ ID No 36, or the sequence SEQ ID No 36 wherein at least one point mutation may occur.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgE heavy chain, when the CH reverse primer has the sequence SEQ ID No42 (HIGCGE4), and when the separated amplifications are real-time separated amplifications, the CH labeled hydrolysis probe has the sequence SEQ ID No43, or the sequence SEQ ID No43 wherein at least one point mutation may occur.
Indeed, for analysis of the IgE repertoire, two couples of primers may be used, namely:
Furthermore, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgE heavy chain and when the separated amplification products obtained for each of the VH subgroups are further elongated, the CH labeled reverse probe has the sequence SEQ ID No 37, or the sequence SEQ ID No 37 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgG heavy chain and when the separated amplifications are real-time separated amplifications, the CH labeled hydrolysis-probe has the sequence SEQ ID No 34, or the sequence SEQ ID No 34 wherein at least one point mutation may occur.
Furthermore, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgG heavy chain and when the separated amplification products obtained for each of the VH subgroups are further elongated, the CH labeled reverse probe has the sequence SEQ ID No 35, or the sequence SEQ ID No 35 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the CH labeled reverse probe SEQ ID No35 is fluorescent on its 5′ part.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgA heavy chain, the CH reverse primer has the sequence SEQ ID No 44, or the sequence SEQ ID No 44 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgA heavy chain and when the separated amplifications are real-time separated amplifications, the CH labeled hydrolysis-probe has the sequence SEQ ID No 46, or the sequence SEQ ID No 46 wherein at least one point mutation may occur.
More preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is an IgA heavy chain and when the separated amplification products obtained for each of the VH subgroups are further elongated, the CH labeled reverse probe has the sequence SEQ ID No 45, or the sequence SEQ ID No 45 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the CH labeled hydrolysis-probes having the sequences SEQ ID No 29, SEQ ID No 34, SEQ ID No 36, SEQ ID No43 and SEQ ID No46 may have 2 point mutations in their sequences.
The point mutations which may occur in the sequences of these CH labeled hydrolysis-probes may be any point mutations provided that this sequence is of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur.
In one further embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that further separated amplifications for each of JH subgroups are performed from the separated amplification products obtained for at least one given VH subgroup of the VH subgroups with the CH reverse primer, said further separated amplifications being performed using a VH internal forward primer corresponding to the given VH subgroup, and associated with a set of JH reverse primers corresponding to the JH subgroups and capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the junction segments of the given type of immunoglobulin heavy chain.
The stringent conditions applied for the further separated amplifications using the VH internal forward primer associated with the set of JH reverse primers are the same as that applied for the amplification step using the set of VH forward primers associated with the CH primer (see supra). Most preferably, the Tm for the amplification step using the VH internal forward primer associated with the set of JH reverse primers is about 60° C.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the further separated amplifications are real-time amplifications performed using a VH labeled forward probe, preferably a VH labeled forward hydrolysis-probe, capable of specifically hybridizing in stringent conditions with the variable segment of the given type of immunoglobulin heavy chain and capable of emitting a detectable signal everytime each amplification cycle occurs, and characterized in that the signal obtained for each JH subgroup is measured.
Among the stringent conditions applied for the VH labeled forward hydrolysis-probe capable of specifically hybridizing with the variable segment of the given type of immunoglobulin heavy chain is the Tm, which is in the range of about 68° C. to about 70° C.; preferably, the Tm for the VH labeled forward hydrolysis-probe is in the range of about 69° C. to about 70° C.; Most preferably, the Tm applied for the VH labeled forward hydrolysis-probe is about 70° C.
More preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given VH subgroup is the VH5 subgroup, the VH5 internal forward primer has the sequence SEQ ID No 31, or the sequence SEQ ID No 31 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that, when the given VH subgroup is the VH4 subgroup, the VH4 internal forward primer has the sequence SEQ ID No 47, or the sequence SEQ ID No 47 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
More preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the VH labeled forward hydrolysis-probe has the sequence SEQ ID No 32, or the sequence SEQ ID No 32 wherein at least one point mutation may occur.
Preferably, the VH labeled forward hydrolysis-probe having the sequence SEQ ID No 32 may have 2 point mutations in its sequence.
The point mutations which may occur in the sequences of this VH labeled forward hydrolysis-probe may be any point mutations provided that this sequence is of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur.
In one further embodiment, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that separated elongations are performed for each of the JH subgroups from the separated amplification products obtained for at least one given VH subgroup of the VH subgroups with the CH reverse primer, said further separated elongations being performed using a set of JH labeled reverse primers corresponding to JH subgroups and capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the junction segments of the given type of immunoglobulin heavy chain, said JH labeled reverse primers being capable of emitting a detectable signal, and characterized in that the elongation products are separated, for each of the JH subgroups, relative to their length, the signal obtained for the separated elongation products is measured, and the quantitative and qualitative profile of the labeling intensity relative to the elongation product length is established, for each of the JH subgroups for the given VH subgroup.
The stringent conditions applied for the set of JH labeled reverse primers used for the elongation and capable of specifically hybridizing with the junction segment of the given type of immunoglobulin heavy chain is the Tm, which is in the range of about 58 and about 60° C., preferably, about 59° C., most preferably, about 60° C.
Preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the set of JH forward primers, optionally labeled, comprises at least the 6 following subgroups of JH primers corresponding to the JH subgroups:
More preferably, the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the sequences SEQ ID No 16 to SEQ ID No 25 may contain at least one to three point mutations, except for the nucleotides 1 to 6 of their 3′ part.
One another subject of the present invention is a set of VH forward primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the variable segments (VH) of immunoglobulin heavy chains, said variable segments being distributed among at least 8 VH subgroups, associated with a CH reverse primer, or a mixture thereof, capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of a given type of an immunoglobulin heavy chain, characterized in that the set of VH forward primers comprises at least the 8 following subgroups of VH primers corresponding to the VH subgroups:
Preferably, the set of VH forward primers according to the present invention is characterized in that the sequences SEQ ID No 1 to SEQ ID No 15 may contain at least one to three point mutations, except for the nucleotides 1 to 6 of their 3′ part.
More preferably, the set of VH forward primers according to the present invention is characterized in that the CH reverse primer is selected from the CH reverse primers capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the constant segments (CH) of the IgM heavy chain, the IgE heavy chain, the IgG heavy chain and the IgA heavy chain.
Preferably, the set of VH forward primers according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgM heavy chain, the CH reverse primer has the sequence SEQ ID No 26, or the sequence SEQ ID No 26 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the set of VH forward primers according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgE heavy chain, the CH reverse primer has the sequence SEQ ID No 33, or the sequence SEQ ID No 33 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Preferably, the set of VH forward primers according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgE heavy chain, the CH reverse primer has the sequence SEQ ID No 42, or the sequence SEQ ID No 42 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
In another preferred embodiment, the set of VH forward primers according to the present invention is characterized in that, when the given type of immunoglobulin heavy chain is the IgG type, a mixture of two CH reverse primers is associated with the set of VH forward primers, said two CH reverse primers having the sequences SEQ ID No 27 and SEQ ID No 28, or the sequences SEQ ID No 27 and SEQ ID No 28 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
More preferably, the set of VH forward primers according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgG heavy chain, the sequence of the CH reverse primer is selected from the group consisting of SEQ ID No40 and SEQ ID No41, or SEQ ID No40 or SEQ ID No41 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of their 3′ part. Preferably, the sequence of the CH reverse primer is SEQ ID No41.
In another preferred embodiment, the set of VH forward primers according to the present invention is characterized in that, when the CH reverse primer is capable of specifically hybridizing in stringent conditions with the nucleic acid encoding the constant segment (CH) of the IgA heavy chain, the CH reverse primer has the sequence SEQ ID No 44, or the sequence SEQ ID No 44 wherein one to three point mutations may occur, except for the nucleotides 1 to 6 of its 3′ part.
Another subject of the present invention is a method for the in vitro diagnosis of a condition associated with an abnormal expression of the repertoire of a given type of an immunoglobulin heavy chain by a lymphocyte B population in a subject, characterized in that it comprises the following steps:
Preferably, the method for the in vitro diagnosis according to the present invention is characterized in that the condition is an auto-immune disease, a B cell lymphoma or an immunodepressive disease.
In another preferred embodiment, the method for the in vitro diagnosis according to the present invention is characterized in that the condition results from a bone marrow transplantation, from a vaccinal test or from an allergic reaction.
Another subject of the present invention is a method for the in vitro follow-up of a treatment of a condition associated with an abnormal expression of the repertoire of a given type of an immunoglobulin heavy chain by a lymphocyte B population in a subject, characterized in that it comprises the following steps:
Preferably, the method for the in vitro follow-up according to the present invention is characterized in that the condition is an auto-immune disease, a B cell lymphoma or an immunodepressive disease.
In another preferred embodiment, the method for the in vitro follow-up according to the present invention is characterized in that the condition results from a bone marrow transplantation, from a vaccinal test or from an allergic reaction.
Another subject of the present invention is a kit for determining the quantitative and qualitative profile of the repertoire a given type of an immunoglobulin heavy chain expressed by a lymphocyte B population present in a tissue sample, characterized in that it comprises the set of VH forward primers according to the present invention associated with the CH reverse primer.
Preferably, the kit according to the present invention is characterized in that it further comprises a set of JH reverse primers, optionally labeled, corresponding to the JH subgroups and capable of specifically hybridizing in stringent conditions with the nucleic acids encoding the junction segments of the given type of immunoglobulin heavy chain.
More preferably, the kit according to the present invention is characterized in that the set of JH reverse primers comprises the 6 following subgroups of JH primers corresponding to the JH subgroups:
More preferably, the kit according to the present invention is characterized in that the sequences SEQ ID No 16 to SEQ ID No 25 may contain at least one to three point mutations, except for the nucleotides 1 to 6 of their 3′ part.
Kits comprising primers according to the present invention are well described in the literature and may further comprise suitable reagents.
Another subject of the present invention is the use of the kit according to the present invention, for the in vitro diagnosis of a condition associated with an abnormal expression of the repertoire of a given type of an immunoglobulin heavy chain by a lymphocyte B population in a subject.
Preferably, the use of the kit according to the present invention is characterized in that the condition is an auto-immune disease, a B cell lymphoma or an immunodepressive disease.
In another preferred embodiment, the use of the kit according to the present invention is characterized in that the condition results from a bone marrow transplantation, from a vaccinal test or from an allergic reaction.
The purpose of the legends of the figures and of the examples below is to illustrate the invention. It doesn't limit the scope of the claimed invention.
In
Clonal expansion A has been found in the course of VH5-JH2 exhaustive sequencing from donor 749. All the sequences from clonal expansion A have the same CDR3 of 14 aminoacids with the sequence SEQ ID No38. Clonal expansion B have been found in the course of VH5-JH1 exhaustive sequencing from donor 749. All the sequences from clonal expansion B have the same CDR3 of 25 aminoacids with the sequence SEQ ID No39. The sequences differs in the mutations occurring in the VH5 region. Codons where mutations occur are initiated in red according to standart nomenclature.* or ** means differents mutations occurring on the same codon. Numbers of each clone for a given pattern of mutations are indicated in black. All mutations occurs from the germline clones (GL). Dashed clones are not found virtual intermediate clones. All existing clones are denominated by a letter.
VH5-JH1 exhaustive sequencing from donor 743 was realized separately in two samples, W and Z containing the same number of B cells. (See Table 4).
100% correspond to the total B lymphocyte IgM, IgE and IgG repertoires.
The patient MM is suffering from atopic eczema, she has been treated by anti-IgE injections. The treatment has begun after the sample MM6. MM15 and MM26 are samples which are under therapy.
100% correspond to IgM+IgE+IgB B cells
100% correspond to IgE and IgG B cells
100% correspond to IgE and IgG B cells
Test of HIGCE4 probe
Consequently, it is possible, using the B Immunoscope technology, to analyse in a qualitative and quantitative manner the B repertoire of an allergic patient and thus to evaluate the efficacy of an anti-allergic treatment.
1. Cell Preparations, Antibodies and Flow Cytometry.
Blood (approximatively 500 ml) from three healthy donors was obtained from the Etablissement Francais du Sang, Necker Enfants Malades Lecourbe, Paris, France. Donor 789 is a 56 year old woman, donor 743 is a 55 year old woman, donor 522 is a 30 year old man. PBMCs were isolated by centrifugation over Ficoll-Paque (Amersham Biosciences AB, Uppsalla, Sweden) in UNI-SEPmaxi+tubes (Novamed, Jerusalem, Israel).
B cells from donor 789 were then obtained from the PBMCs by depletion using the B cell negative isolation kit from Dynal (Dynal, Oslo , Norway). For donors 743 and 522, PBMCs were co-stained with CD19 beads and CD19-PE (Pharmingen, San Jose, Calif.) and purified by positive selection on AutoMACS using respectively Possel or Possel-S programs (Miltenyi Biotec, Bergisch-Gladbach, Germany). The eluted cells from the positive and negative fractions, as well as the total PBMCs were then labeled with either anti-IgM, anti-IgG, anti-IgD, Ig-E, anti-IgA1/IgA2, anti-kappa and anti-lambda antibodies (Pharmingen, San Jose, Calif.) and rabbit polyclonal anti-Human IgM (Jackson Immunoresearch laboratories, West Grove, Pa.).
Using these different antibodies, the percentage and the absolute number of cells bearing the different antibody isotypes were determined among the PBMC and B cells positive fraction of donor 743 and 522 (Table 1). The purity of the positive fraction was checked either by the CD19 staining, either by the sum of anti-kappa and anti-lambda staining.
2. RNA and cDNA Preparation
Cells from both positive and negative fractions were aliquoted and kept frozen at −20° in the lysis buffer of the RNeasy mini-kit (Qiagen, Courtaboeuf, France). Total RNA was extracted using the RNeasy mini-kit (Qiagen, Courtaboeuf, France) according to the manufacturers specifications, as previously described (Lim and al. (2002). J Immunol Methods 261, 177-94). cDNA was then prepared by RNA reverse-transcription with 0,5 μg/μl oligo (dT) 17 and 200 U of Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France).
3. Quantitative Immunoscope
Quantitative Immunoscope analysis was performed as described (Lim and al. (2002). The different VH germline genes can be clustered in seven families according to their level of homology. Both IMGT (http://imgt.cines.fr) (Lefranc and al. (1999). Nucleic Acids Res 27, 209-12) and Vbase databases (http://www.mrc-cpe.cam.ac.uk) (Tomlinson and al. (1998). V Base Sequence Directory. In, M. C. f. P. Engineering, ed. (Cambridge, UK.)) were used to have access to their sequences and the germinal gene were aligned using GCG Wisconsin package program (http://www.accelrys.com/products/gcg_wisconsin_package).
All the primers used are shown in Table 2. In order to have the opportunity to study somatic mutations in the VH regions, VH family specific primers were chosen in the FR1 region for all the VH families except VH3 and VH4 families where the specific primers were designed in the FR3 region. VH3 gene family, the largest one, was divided in two sub-families VH3a and VH3b in order to achieve a better specifity. This specifity was tested by specifically amplify each VH family from a PBMC mix of three heathly donors using VH family specific primers and HIGCM primer, PCR products cloning and sequencing. For only one VH family, the VH7 family, full specifity of the amplification was not achieved as few of the amplified VH were belonging to the VH1 family.
For the quantification of the VH families usage for IgM expressing cells, an aliquot of the cDNA was amplified using pfu high fidelity Taq polymerase (Stratagene, Amsterdam, The Netherlands) with each of the 8 families VH specific primers on one side, the HIGCM primer on the other side and the TM-MGB-HCM probe, a Taqman Minor Groove Binder (MGB) FAM-labeled nested probe specific for the Cμ region.
For the quantification of the JH usage, an aliquot of the cDNA was amplified on the 5′ side with the VH5int specific primer (SEQ ID No 31) and each of the 6 JH specific primers on the 3′ side together with the TM-MGB-VH5int probe (SEQ ID No 32), a Taqman MGB FAM-labeled nested probe specific for the VH5 family. All Taqman MGB probes were designed using the Primer express software program (Applied Biosystems, Courtaboeuf, France). PCR reactions were carried out in 25 μl total volume. For all these differents reactions, real time quantitative PCR was then performed on an ABI 5700 system (Applied Biosystems, Courtaboeuf, France). The relative usage of each VH family or each JH segment was calculated according to the above formula:
In which Ct(x) is the fluorescent threshold cycle number measured for VH(y) family or JH(y). In this case, the VH family primers pair display a mean efficiency of 0.95±0.08 and the JH segment primers display a mean efficiency of 0.91±0.08.
For the VH family amplifications, 2 μl of each amplification reactions were used as template in a run-off reaction initiated by either the HCM-FAM primer, a fluorescent nested Cμ primer, the JH1-FAM: 5′-Fam-CCCTGGCCCCAGTGCTG-3′ (SEQ ID No 16) or JH2-FAM 5′Fam-CCACGGCCCCAGAGATCG-3′ (SEQ ID No 17) primers or the VH5-FAM primer in a total volume of 10 μl as previously described (Pannetier and al. (1997). In The Antigen T Cell receptor: Selected Protocols and Applications., J. R. E. Oksenberg, ed. (Landes Bioscience, Chapman & Hall), pp. 287). All fluorescent fragments were then separated on a denaturing 6% acrylamide gel, run on an automated 373 DNA sequencer (Applied Biosystems, Courtaboeuf, France) and analyzed with Immunoscope software (Pannetier and al. (1993). Proc Natl Acad Sci U S A 90, 4319-23).
4. CDR3 Band Purification
In the case of VH5-JH2 rearrangement amplification, bands corresponding to a CDR3 length of 8 amino-acids had to be purified from the acrylamide gel. For this purpose, the PCR products were made visible on the acrylamide gel by a DNA silver staining system as previously described (Promega, Madison, Wis.) (Casrouge and al. (2000). J Immunol 164, 5782-7; Raaphorst and al. (1996). Biotechniques 20, 78-82, 84, 86-7). The bands of interest were cutted from the gel and disrupted in 50 μl of TE. A second 33 cycles PCR using the VH5int and the JH2 primers was then conducted with 1 μl of the band purification recovered PCR product and the amplification was then cloned as follow.
5. VH Transcript Sequencing and Analysis
Either the total product of the VH5-JH1 rearrangement from different donors or the two PCR bands purified products amplified from the VH5-JH2 (8aa) CDR3 bands) were cloned using the TOPO Blunt PCR cloning kit (Invitrogen, Cergy Pontoise, France). Sequencing reactions were performed as previously described (Arstila and al. (1999). Science 286, 958-61) directly on these products using VH5int primer and the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Courtaboeuf, France). These sequencing reactions were then run on an ABI PRISM 3700 DNA analyser (Applied Biosystems, Courtaboeuf, France). CDR3 regions and VH mutations of the corresponding sequences were extracted and analysed using Taps1.1 software written by Emmanuel Beaudoing (Casrouge and al. (2000). J Immunol 164, 5782-7).
6. Calculation of the VH/Bcells Repertoire Size
The size of the VH repertoire can be estimated to equal the number of distinct sequences found for a given rearrangement (when sequencing is undergone to saturation) divided by the product of the considered VH frequency with the considered JH frequency. If the size of the repertoire was deduced from a purified CDR3 band, the percentage of this band among the all rearrangement was taken in account in the above calculations. Diversity due to CDR3 variability can be distinguished from the diversity due to somatic mutations according to the way the sequences are analysed on the Taps software. The calculations can be done either with the number of distincts sequences given by limits flanking the CDR3 regions, or including most of the VH region.
In order to estimate the size of the peripheral human B cell repertoire, we have adapted the Immunoscope method developed in our laboratory and previously applied for T cell repertoire studies (Pannetier and al. (1993). Proc Natl Acad Sci U S A 90, 4319-23). In addition, a precise quantification of VH and JH gene usage was performed by coupling real time PCR with Immunoscope analyses. B cells from several donors were subjected to those studies. Table 1 shows representative Facs analysis of two donors, before and after B cells enrichment. cDNA prepared from those cells of two donors were then submitted to a step of PCR amplification using VH family specific primers on one side and one Cμ primer, specific for the constant region of IgM on the other side, together with the TM-MGB-HCM fluorescent probe, nested in the constant region close to the Cμ primer (see Material & Methods and Table 2). Real time determination of the level of fluorescence at each PCR step makes possible the quantification of VH family usage as shown in Table 3a. For a given donor, very large difference of VH family usage could be evidenced, VH3 and VH4 gene families representing the majority of all rearrangements (60% and 20% respectively). This is compatible with a utilisation of VH gene proportional to the complexity of each family, since 27 different genes belong to the VH3 gene family and 10 to the VH4 family. Those results are also in accordance with what have been already determined by single cell PCR (54% of VH3 rearrangement and 23% of VH4 rearrangement out of 491 analysed cells) (Odendahl and al. (2000). J Immunol 165, 5970-9) although they are derived from a much larger cell number (about 70 000 cells). In order to evaluate the reproducibility of the quantification, purified IgM positive B cells from donor 743 were divided into 6 samples (2.7 106 IgM positive cells/sample, see Table 3A). Two of these samples, sample W and sample Z were then tested for VH family usage quantification. As can be seen from Table 3a, VH usage of samples W and Z are almost identical, as expected. In addition, the values obtained for both samples are very similar to those obtained for donor 789, although individual variations could be detected, for example in the VH2 family gene usage.
The inventors then quantified of the JH usage for one given VH family. VH5 family was chosen for two main reasons. 1°) according to IMGT, the number of individual genes is limited to only two germline encoded genes, VH5 and VH5.51. 2°) data on VH gene family usage (Table3A) have shown that VH5 is not overused (1% of all rearrangement in sample W and Z from donor 743 and 3.2% for donor 789). For the quantification of JH gene utilisation, VH5-Cμ amplification PCR products from the two different donors were subjected to a second PCR amplification using VH5int primer specific for the VH5 genes on one side and each of the 6 JH specific primers on the other side, together with the TM-MGB-VH5int fluorescent probe, nested in the vicinity of the VH5int primer and specific for the VH5 genes (see Material & Methods and Table 2). As shown in Table 3B, overutilisation of JH4 was observed for both donors. Again, separate quantification of samples W and Z from donor 743 gave almost identical results. Nevertheless, overall JH usage was less similar when donors 789 and 743 were compared (Table 3B). For the rest of thes study, the inventors have focused two gene segments, JH1 and JH2, because they are the less used in all rearrangements of both donors 789 and 743.
Following quantification of VH and JH usage, PCR amplification was submitted to a <<run-off >> reaction, as previously described (Pannetier and al. (1993). Proc Natl Acad Sci U S A 90, 4319-23].
To estimate the global size of the B cell repertoire, the inventors first focused on the VH5-JH2 rearrangement in donor 789 for the following reasons. First, VH5 family only includes two germline genes and its overall usage in the constitution of the antibody repertoire is not more then 3.2% (see Table 3). Second, JH2 segment is the second less utilized gene segment among all JH fragments (2,1% of all VH5 rearrangements) (Table 3). Therefore, it can be hypothesised that, after Immunoscope separation and quantification of all CDR3 peaks, the exhaustive sequencing of those rearrangements will be feasible. Third, the Immunoscope profile of VH5-JH2 rearrangement in donor 789 is representative of most blood B cell rearrangements, characterised by a quasi-Gaussian repartition only slightly perturbed by the presence of few clonal expansions (
The first objective was to study the general size of the B cell repertoire irrespectively of the isotype expressed by the antibody produced. To achieve this goal, cDNA from donor 789 was PCR amplified using VH5 gene family-specific primer together with IGJH2 specific primer (see Table 2). <<Run off>> reaction was then performed with a JH2-Fam fluorescent probe and the specific Immunoscope profile obtained (
As IgM positive B cells are by far the most frequent subset among PBMC (Table 1), the inventors then focused on the determination of repertoire size of these cells. For this purpose, the inventors decided to concentrate on VH5-JH1 rearrangement because on one hand, JH1 gene segment is the less utilized among all JH fragments (0.2%) (Table 3) and on the other hand exhaustive sequencing was possible on the whole VH5-JH1 rearrangement. In this respect, cDNA from donor 789 was PCR amplified using the VH gene family and the Cμ specific primers, followed by “run-off” reaction using a VH5-Fam fluorescent probe (Table 2). The specific VH5-JH1 immunoscope profile is shown in
To confirm this finding and to validate of this approach, the inventors performed global sequencing of the VH5-JH1 rearrangement amplified from donor 743, and this was done twice on the two samples W and Z, which harbor the same number of IgM positive B cells (2.7 106 i.e. ⅙ of the total amount of purified B cell). The specific VH5-JH1 immunoscope is shown in
As compared with T cells, one of the most striking difference in generating diversity in B cells is the property to accumulate somatic mutations in their variable regions. Somatic mutations are supposed to be generated mainly in the germinal center, as the key phenomenon in affinity maturation and maintenance of memory (Wu and al. (2003). J Clin Immunol 23, 235-46). Somatic mutations are not restricted to IgG expressing B cells since 35% of human blood IgM+B cells have been reported to display somatic mutations in heavy chain variable genes (Klein and al. (1997). Blood 89, 1288-98). The calculations described above to define the size of the B cells repertoire take into consideration CDR3 diversity, but not the diversity generated by somatic mutations. The inventors studied first these mutations that were detected in two clones during the exhaustive sequencing of the VH5-JH2 and VH5-JH1 rearrangements. By definition, the inventors consider that belong to the same clone all the sequences bearing the same CDR3. Using this criteria, it is possible to draw phylogenic trees of the mutations. Clone A originates from the sequencing of VH5-JH2 rearrangement from donor 789, it bears a CDR3 sequence of 14 aminoacids, it expresses an IgM receptor and has been found 67 times. Clone B originates from the sequencing of VH5-JH1 rearrangement from donor 743, it bears a CDR3 sequence of 25 aminoacids, i expresses an IgM receptor and have been found in 130 sequences. Both clone A and B mutations trees are shown in
If those two examples of clonal mutations can be informative in the follow-up of a given immune response, they are not significant enough to evaluate the contribution of somatic mutations to the diversity. In order to calculate the global contribution of somatic mutations to diversity, the inventors have compared all the VH sequences obtained from exhaustive sequencing of the different rearrangements to the germline VH irrespectively of the CDR3 sequences. Results are shown in Table4. For both donors and for both VH5-JH2 and VH5-JH1 rearrangements, approximately half of the VH sequences are found bearing one or several somatic mutations. Taking into account this B cell specific additional source of diversity, the global repertoire size of the studied blood samples can be estimated (Table 4). For donor 789, the value obtained for 500 ml blood sample ranges from 6.7×106 to 107 depending of the rearrangement studied, the methodology used (with or without band purification), and the specificity of the PCR amplification (all isotypes or only IgM). Taking into account a degree of uncertainty inherent to the approach used, the VH repertoire of B cell repertoire is around one order of magnitude larger than the Vβ T cells repertoire (Arstila and al. (1999). Science 286, 958-61).
This result has other implications in what concern the properties of the B cells repertoire. The estimated size of the B cell repertoire, calculated from a given number of B cells, is always very close to this number and this was even more manifest when only a small number of B cells was studied, as in W and Z samples from donor 743. In this case, the repertoire number obtained was even slightly larger than the number of B cells, but still included the confidence limits interval (see Table 3). In other words, in a 500 ml blood poach, according to the diversity of their VH genes repertoire, each B cells express and produce a different antibody, with the exception of antigen specific clonal expansions. Consequently, assuming that the total amount of blood in one given individual is close to 5 liters, the global B cells clone size must be comprised between 10 and 1 and therefore the diversity of B cell repertoire must range accordingly between 107 and 108. Again, this result strengthens the differences existing between T cells and B cells repertoire as the T cell Vβ clone size has been reported to be approximatively equal to 100 (Arstila and al. (1999). Science 286, 958-61).
Another property of B cells is the capability of developing very large clonal expansions in response to antigenic stimulation. Clonal expansion can be defined according to different criteria: 1°), it corresponds to all sequences sharing the same CDR3 sequence, irrespective on the presence or not of somatic mutations in their V regions. The inventors have used this definition to represent trees of mutations for a given clone (see
1°) The Diversity of the Immunoglobulin VH Genes Repertoire is Comprised Between 107 and 108 Different Expressed Rearrangements
In this study, the inventors report the first size estimate of the human B cells VH gene repertoire. To reach this goal, the inventors first quantified by real time PCR VH gene utilisation in total peripheral B cells isolated from normal donors. The inventors then focus on the VH5 gene family which display an intermediate rate of utilisation and is composed by only few germline genes. JH gene segment utilisation was then quantified in all VH5 gene family rearrangements. Two rearrangements, VH5-JH1 and VH5-JH2, were chosen and extensively studied. In the case of VH5-JH1 rearrangement, Immunoscope was performed in order to separate and quantify the representation of 8 and 9 aminoacids long CDR3. The corresponding bands were then purified, cloned and subjected to exhaustive sequencing. Calculations have allowed to estimate the size of the VH gene repertoire. For the VH5-JH2 rearrangement, exhaustive sequencing was performed on the total PCR product, for all the different CDR3 lengths. In both case, the estimate of the VH gene global diversity is close to 107 for all B cells purified from 500 ml of human blood. If the total amount of blood of a normal donor is roughly equal to 5 liters, the global diversity of the human peripheral B cell VH gene repertoire must be close to 108 expressed rearrangements
2°) The Mean Global Clone Size of Peripheral B Cells is Close to 1
From the above result on global diversity of the human peripheral B cell, it can be estimated that the global clone size of immunoglobulin expressing cells must varied between 1 and 10. As a matter of fact, when separated sequencing is performed on VH5-JH2 rearrangement from two samples of the same donor harboring the same number of cells, the size of the repertoire calculated in both case is almost identical to this number of cells. The same result was obtained with different number of cell in the studied sample, even when the number of cells is equal to the B cells total number of the blood gift as for the sequencing of VH5-JH1 rearrangement In addition, a detained study of the sequences of each of this two samples show that, with the exception of large clonal expansions with mutated VH genes, not more than 5% of the sequences are found in both samples. Therefore, if B cell diversity from a blood gift can be estimated to reach 107 different VH and if the mean global clone size for B cell is close to 1, the total VH diversity must reach 108. Alternatively, if B cell mean global clone size is closer to 10, the overall size of the VH repertoire of the donor should be similar to that of the blood gift, i.e. 107.
3°) VH-VL Pairing is Poorly Involved the Generation of B Cells Diversity
If the calculation of the diversity of VH repertoire always give a number similar to that of the number of cells studied, one can assume that VH genes diversity can be similar to B cells diversity. On the contrary than for the T cells repertoire, VL repertoire and VH-VL pairing are less involved in the diversity of the B cells repertoire than Vα repertoire and Vα-Vβ paring in the diversity of T cells repertoire. If each B cells in the periphery which cannot be considered to belong to a clonal expansion (and therefore representing at least 70% of all B cells) is already identify simply according to its VH usage, size determination of the VL repertoire can be assumed to provide similar values than for VH calculations. In other words, if VH rearrangement is enough by itself to define a given B cell, each B cell expressing a particular VH-VL pair is necessarily single. This situation is completely different to what have been already reported for the T cells repertoire, where each VB must pair in average with 25 different α chain leading to a total αβ TCR diversity in the blood not less than 25×106 differents TCR. Therefore, if both T and B cells repertoire reach approximatively the same size of repertoire (between 107 and 108 different expressed rearrangements), the way by which this level of diversity is achieved strongly differ for each cell type. In B cells, the size of the repertoire mainly rely on VH CDR3 diversity and on VH somatic mutations. Consecutively, the size of the VH repertoire is at least one order of magnitude larger as compared to the Vβ repertoire. On the contrary, in addition to Vβ and Vα diversity, T cells repertoire diversity result also in the different possibilities of pairing between Vα and Vβ chains. The importance of Vα-Vβ pairing in the T cells repertoire diversity have been postulated to result in part from several rounds of cell divisions between Vβ and Vα rearrangements, allowing the same Vβ to pair with differents Vα. This rise the possibility that VL rearrangements could occur in B cells shortly after VH rearrangements within a window of time allowing less cell divisions than for T cells, preventing a B cell clone expressing a given VH to expand as much as a T cell clone. Evidences showing that, in the mouse, VL rearrangements can even occur prior to VH rearrangements are also in line with this hypothesis. The importance of Vα-Vβ pairing in the T cell repertoire diversity can also derived from thymocyte positive and negative selection in the thymus. The result of this process lead to the induction of apoptosis for a large majority of cells and selection of a small number according to the specificity of their TCR. Therefore, blood circulating T might be more representative of a Vα-Vβ pair selected population while blood circulating B cells, with the exception of clonal expansions, could be more closely related to the bone marrow outcome. For the T cells, the selection of the Vα-Vβ pair occur in the thymus on the basis of MHC recognition, and allow the local expansion of the cells expressing the selected TCR. Indeed, the mean clone size for T cell have been estimated to reach 100 copies in the periphery. For B cells, on the contrary, our results show a much smaller clone size, ranging between 1 and 10. Another hypothesis to explain this difference, would be to postulate that bone marrow produce B cells with fewer constraints of selection than for T cells in the thymus. Indeed, two major steps of selection of B cells have been reported to occur in the bone marrow. First, once a functional VDJ rearrangement is achieved and an μ heavy chain expressed at the surface of the pre-B cell, failure of pairing with the VpreB lambda5 surrogate light chain could prevent further differentiation. Second, cells expressing a self reactive BCR have been described to modify their receptor specificity by a process denominated <<receptor editing>>. Nevertheless, the properties of BCR antigen recognition, leading to the ability to identify a three dimentional pattern, differ strongly with the TCR capacity to recognize a given peptide in the context of a given MHC. In addition, our results being consistent with no or few antigen independent cell divisions in the bone marrow, its outcome could be more representative of a random distribution of BCR than the thymus TCR outcome. Indeed, being deduced from total peripheral B cells, our results do not allow the complete assimilation of this population with the bone marrow outcome. Furthermore, in the mouse, peripheral B cells have been described to be mainly ligand selected . In order to gain some insights on this issue, studies on purified B cell populations representative of the recent bone marrow emigrant should be informative.
4°) Clonal Expansions Account for 20 to 30% of a Given Rearrangement
Over than 15 years ago, Langman and Cohn have proposed, based on informatic stimulations and on B cells repertoire observations that B cell repertoire could be divided in several identical sub-fractions, each sub-fraction being a <<functional unit>> of 107 B lymphocytes bearing a repertoire of 105 rearrangements (Langman and Cohn, 1987). Our findings that the global B cell clone size must be be comprise between one and ten and that most peripheral B cells are unique are apparently incompatible with the protection theory which postulate, in organisms harboring more than 107 lymphocytes, a important redundancy of BCR specificities. Paradoxically, T cells repertoire properties, as they were previously described (Arstila et Wagner) seems more compatible with a direct interpretation of the protection theory. First, as already mentioned by Casrouge et al, the difference of T cells diversity between mice [Casrouge and al. (2000). J Immunol 164, 5782-7]and human [Arstila and al. (1999). Science 286, 958-61]does not reflect the much larger difference in total number of peripheral T lymphocytes between these two species. Second, T cells global clone size appear, in particular in human, to be much larger than B cells one and therefore being compatible with the existence of several functional units.
Another property of the B cells repertoire hardly seems compatible with our results. In both mice and human, B cells produce <<natural antibodies>> which are mainly low affinity IgM antibodies, usually polyspecific and germ line encoded. Those antibodies, which are predominant in newborn life, often recognise self antigen. Their production is constant over life period and very stable, antibodies against given specificities always being found in differents individuals. How could this property of the B cells repertoire could be achieved if almost each peripheral B cell habor a unique specificity?
In order to reconcile our findings with both protection hypothesis and production of natural antibodies, the inventors wishes to propound the following hypothesis. The maintenance of natural antibody production could be achieved, not by a continuous bone marrow outcome, but rather by continuously activated peripheral, B cells, self ligand selected and eventually giving rise to clonal expansions. Similarly, protection theory could only concern the pool of clonal expansions among all peripheral B cells. In this case, the stability of natural antibodies production will be ensure mainly by peripheral proliferation of clonal expansions, which represent, according to our results, up to 30% of the total number of sequences for one given rearrangement. As illustrated in the two examples of clonal expansions described in
The results provide therefore, in addition to the first determination of the size of the peripheral B cells repertoire, some hypothesis for a better understanding of the mechanisms involved in the maintenance of this repertoire. The quantitative approach developed here could also be a powerfull tool to study B cells repertoire disturbancies occurring during pathological and autoimmune diseases.
Table 1: Proportion and Absolute Numbers of Cells Expressing Different Isotype in the Blood of Two Healthy Donors
Blood cells from donor 743 and 522 were stained before and after B cell enrichment with the different antibodies according to the material and method and analysed on a FACScalibur (Becton Dickinson). For each staining, result are expressed in % of total cells and corresponding absolute numbers are shown in parenthesis.
Table 2: Specific Primers for B Cell Heavy Chain Quantitative Immunoscope Analysis
This table shows the list of primers utilized to quantified VH and JH utilization by real time PCR. Each primers specifically amplify one or several VH, as notified. The primers localisation in the FR1 or FR3 is also shown. 3′ end phosphorothioate chemical modification of some primers is mentioned by a *. Only two primers display degenerate sites with R meaning A or G and S meaning G or C.
Table 3: VH and JH Quantification by Real Time PCR
Table 3 A shows the mean percentage of VH utilisation determined by real time PCR for B cells from donor 789 and 743. For donor 743, this determination was done separately on the two samples W and Z, as displayed. Table 3B shows the JH segment usage in association with the VH5 gene family.
| Number | Date | Country | Kind |
|---|---|---|---|
| 03293159.4 | Dec 2003 | EP | regional |
| 10/734,622 | Dec 2003 | US | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB04/04413 | 12/15/2004 | WO | 11/28/2006 |
