# Patent

## Title
Method for evaluating and comparing immunorepertoires

## Abstract

Disclosed is a method for amplifying RNA and/or DNA from immune cell populations and using the amplified products to produce an immune response profile and evaluate the possible correlation between a normal or abnormal immune response and the development of a disease such as an autoimmune disease, cancer, diabetes, or heart disease.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority of U.S. provisional patent application No. 61/045,586, filed Apr. 16, 2008.


FIELD OF THE INVENTION
The invention relates to methods for identifying biomarkers and to methods for identifying T-cell receptor, antibody, and MHC rearrangements in a population of cells.
BACKGROUND OF THE INVENTION
Scientists have known for a number of years that certain diseases are associated with particular genes or genetic mutations. Genetic causation, however, accounts for only a portion of the diseases diagnosed in humans. Many diseases appear to be linked in some way to the immune system's response to infectious and environmental agents, but how the immune system plays a role in diseases such as cancer, Alzheimer's, costochondritis, fibromyalgia, lupus, and other diseases is still being determined.
The human genome comprises a total number of 567-588 Ig (immunoglobulin) and TR (T cell receptor) genes (339-354 Ig and 228-234 TR) per haploid genome, localized in the 7 major loci. They comprise 405-418 V, 32 D, 105-109 J and 25-29 C genes. The number of functional Ig and TR genes is 321-353 per haploid genome. They comprise 187-216 V, 28 D, 86-88 J and 20-21 C genes (http://imgt.cines.fr). Through rearrangement of these genes, it has been estimated that approximately 2.5×107 possible antibodies or T cell receptors can be generated.
Although, at the germine level, human beings are capable of generating large numbers of diverse Igs and TRs, the number of available Igs and TRs for a particular individual is actually much smaller due to negative selection during B and T cell development. In some individuals, this process may not remove some of the cells that would cross-react with the body's own tissues, and this may be the cause of some types of autoimmune diseases.
A few diseases to date have been associated with the body's reaction to a common antigen (Prinz, J. et al., Eur. J. Immunol. (1999) 29(10): 3360-3368, “Selection of Conserved TCR VDJ Rearrangements in Chronic Psoriatic Plaques Indicates a Common Antigen in Psoriasis Vulgaris”) and/or to specific VDJ rearrangements (Tamaru, J. et al., Blood (1994) 84(3): 708-715, “Hodgkin's Disease with a B-cell Phenotype Often Shows a VDJ Rearrangement and Somatic Mutations in the VH Genes”). What is needed is a better method for evaluating changes in human immune response cells and associating those changes with specific diseases.
SUMMARY OF THE INVENTION
The invention relates to a method for producing an immune status profile (ISP) for a human and/or animal. In one aspect of the invention, the method comprises the steps of amplifying, in a first amplification reaction using target-specific primers, at least one RNA and/or DNA from a sample of white blood cells from at least one human or animal subject to produce at least one amplicon, at least a portion of the target-specific primers comprising additional nucleotides to incorporate into a resulting amplicon a binding site for a common primer; rescuing the at least one amplicon from the first amplification reaction; amplifying, by the addition of common primers in a second amplification reaction, the amplicons of the first amplification reaction having at least one binding site for a common primer; and sequencing the amplicons of the second amplification reaction to identify and quantify DNA sequences representing antibody and/or receptor rearrangements to create an immune status profile.
In another aspect of the invention, the step of rescuing the at least one amplicon from the first amplification reaction may be omitted, and the first and second amplification reactions may occur without separation of the amplicons from the target-specific primers. Genomic DNA may also be amplified, and the step of amplifying DNA may be substituted for the step of amplifying RNA, especially in cases where analysis of an immune system component such as the major histocompatibility complex (MHC) is desired.
In aspects of the invention, subpopulations of white blood cells may be isolated by flow cytometry to separate naïve B cells, mature B cells, memory B cells, naïve T cells, mature T cells, and memory T cells. In various aspects of the method, recombinations in the subpopulation of cells are rearrangements of B-cell immunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK, IgL), T-cell receptor Beta, Gamma, Delta, and/or Major Histocompatibility Complex (MHC) molecules I or II.
In another aspect of the invention, the method may also comprise compiling and comparing the immune cell profile for a population of normal individuals with the immune cell profile for a population of individuals who have been diagnosed with a disease to determine if there is a correlation between a specific rearrangement or set of rearrangements and the disease.
In another aspect of the invention, the method may comprise comparing the immune cell profile identified for a population of individuals to whom a vaccine has been administered with the immune cell profile for a population of individuals to whom the vaccine was not administered to evaluate the efficacy of the vaccine in producing an immune response.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a and FIG. 1b are photographs of gels illustrating the presence of amplification products obtained by the method of the invention using primers disclosed herein.
FIG. 2 illustrates distributions of domain usage for (a) Ig heavy chain in healthy control sample, (b) TCR beta chain in a blood sample from a patient with colon cancer, (c) Ig kappa chain in a blood sample from a patient with Chronic Lymphocytic Leukemia (CLL), and (d) Ig lambda chain in a blood sample from a patient with Systemic lupus erythematosus (SLE). Empty spots indicate missing sequences associated with corresponding V-J combinations and the height of the column indicates the frequency of occurrence of a particular sequence.



DETAILED DESCRIPTION
The inventor has developed a method for evaluating antibody and receptor rearrangements from a large number of cells, the method being useful for comparing rearrangements identified in populations of individuals to determine whether there is a correlation between a specific rearrangement or set of rearrangements and a disease, or certain symptoms of a disease. The method is also useful for establishing a history of the immune response of an individual or individuals in response to infectious and/or environmental agents, as well as for evaluating the efficacy of vaccines.
The invention relates to a method for producing an immune status profile (ISP) for a human and/or animal. In one aspect of the invention, the method comprises the steps of amplifying, in a first amplification reaction using target-specific primers, at least one RNA from a sample of white blood cells from at least one human or animal subject to produce at least one amplicon, at least a portion of the target-specific primers comprising additional nucleotides to incorporate into a resulting amplicon a binding site for a common primer; rescuing the at least one amplicon from the first amplification reaction; amplifying, by the addition of common primers in a second amplification reaction, the amplicons of the first amplification reaction having at least one binding site for a common primer; and sequencing the amplicons of the second amplification reaction to identify and quantify DNA sequences representing antibody and/or receptor rearrangements to create an immune status profile.
Where the term “comprising” is used herein, “consisting essentially of” and “consisting of” may also be used. The term “immune status profile” is intended to mean a profile for an individual or population of individuals indicating the presence and/or absence of sequences representing specific rearrangements representing the diversity of B cells, T cells, and/or other cells of the human and/or animal immune system, as well as the frequency of their occurrence. Where amplicons are referred to as “rescued” herein, it is to be understood that amplicon rescue may occur by the separation of amplicons from the primers which are used to create them, or may occur by dilution of the amplicon/primer mix so that, by virtue of the fact that there are significantly more amplicons than primers from a first amplification reaction, the effect of those primers is minimized in a second amplification reaction using different primers. “Common primers” are those primers that may be used to amplify polynucleotides (e.g., amplicons from a first amplification produced by target-specific primers) having non-identical sequences in general, but sharing sequence similarities in that they contain binding sites for the same primers. Common primers are generally chosen for their efficiency at priming successful amplifications, so their use is effective for achieving higher levels of amplification in a non-target-specific manner in the method of the present invention. Common primer binding sites may be incorporated into amplicons resulting from a first amplification by attaching their sequences or their complementary sequences to the sequence of a target-specific primer. Common primers may be chosen by one of skill in the art by a variety of primer-design methods.
Subpopulations of white blood cells may be isolated by flow cytometry to separate naïve B cells, mature B cells, memory B cells, naïve T cells, mature T cells, and memory T cells. Recombinations in these subpopulations of cells are generally rearrangements of B-cell immunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK, IgL), T-cell receptor Beta, Gamma, Delta, and/or Major Histocompatibility Complex (MHC) molecules I or II.
By performing an additional step, namely that of compiling and comparing the average immune status profile for a population of normal individuals with an average immune status profile for a population of individuals who have been diagnosed with a disease, it is possible to use the immune cell profile to determine if there is a correlation between a specific rearrangement or set of rearrangements and the disease.
The invention also provides a method for evaluating vaccine efficacy, in terms of creating a change in the immune cell profile, by performing the steps of the method and comparing the immune cell profile identified for a population of individuals to whom a vaccine has been administered with the immune cell profile for a population of individuals to whom the vaccine was not administered to evaluate the efficacy of the vaccine in producing an immune response.
In one embodiment of the invention, a peripheral blood sample is taken from a patient and isolation of a subpopulation of white blood cells may be performed by flow cytometry to separate naïve B cells, mature B cells, memory B cells, naïve T cells, mature T cells, and memory T cells. In various embodiments of the method, recombinations in the subpopulation of cells may comprise rearrangements of B-cell immunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK, IgL), T-cell receptor Beta, Gamma, Delta, or Major Histocompatibility Complex (MHC) molecules I or II.
In some aspects, the step of rescuing the amplicons from the first amplification reaction may be omitted and the two amplification reactions may be performed in the same reaction tube without amplicon rescue or dilution of the primers remaining from the first amplification reaction.
The inventor previously developed a PCR method known as tem-PCR, which has been described in publication number WO2005/038039. More recently, the inventor has developed a method called amplicon rescue multiplex polymerase chain reaction (arm-PCR), which is described in U.S. PCT/US09/39552 and herein. Both the tem-PCR and arm-PCR methods provide semi-quantitative amplification of multiple polynucleotides in one reaction. Additionally, arm-PCR provides added sensitivity. Both provide the ability to amplify multiple polynucleotides in one reaction, which is beneficial in the present method because the repertoire of various T and B cells, for example, is so large. The addition of a common primer binding site in the amplification reaction, and the subsequent amplification of target molecules using common primers, gives a quantitative, or semi-quantitative result—making it possible to determine the relative amounts of the cells comprising various rearrangements within a patient blood sample. Clonal expansion due to recognition of antigen results in a larger population of cells which recognize that antigen, and evaluating cells by their relative numbers provides a method for determining whether an antigen exposure has influenced expansion of antibody-producing B cells or receptor-bearing T cells. This is helpful for evaluating whether there may be a particular population of cells that is prevalent in individuals who have been diagnosed with a particular disease, for example, and may be especially helpful in evaluating whether or not a vaccine has achieved the desired immune response in individuals to whom the vaccine has been given.
There are several commercially available high throughput sequencing technologies, such as Roche Life Sciences 454 Sequencing®. In this sequencing method, 454A and 454B primers are either linked onto PCR products during PCR or ligated on after the PCR reaction. When done in conjunction with tem-PCR or arm-PCR, 454A and 454B primers may be used as common primers in the amplification reactions. PCR products, usually a mixture of different sequences, are diluted to about 200 copies per μl. In an “emulsion PCR” reaction, (a semisolid gel like environment) the diluted PCR products are amplified by primers (454A or 454B) on the surface of the microbeads. Because the PCR templates are so dilute, usually only one bead is adjacent to one template, and confined in the semisolid environment, amplification only occurs on and around the beads. The beads are then eluted and put onto a plate with specially designed wells. Each well can only hold one bead. Reagents are then added into the wells to carry out pyrosequencing. A fiber-optic detector may be used to read the sequencing reaction from each well and the data is collected in parallel by a computer. One such high throughput reaction could generate up to 60 million reads (60 million beads) and each read can generate about 300 bp sequences.
One aspect of the invention involves the development of a database of immune status profiles, or “personal immunorepertoires” (PIRs), so that each individual may establish a baseline and follow the development of immune responses to antigens, both known and unknown, over a period of years. This information may, if information is gathered from a large number of individuals, provide an epidemiological database that will produce valuable information, particularly in regard to the development of those diseases such as cancer and heart disease which are thought to often arise from exposure to viral or other infectious agents, many of which have as yet been unidentified. One particularly important use for the method of the invention enables studies of children to determine whether infectious disease, environmental agents, or vaccines may be the cause of autism. For example, many have postulated that vaccine administration may trigger the development of autism. However, many also attribute that potential correlation to the use of agents such as thimerosol in the vaccine, and studies have demonstrated that thimerosol does not appear to be a causative agent of the disease. There is still speculation that the development of cocktail vaccines has correlated with the rise in the number of cases of autism, however, but gathering data to evaluate a potential causal connection for multiple antigens is extremely difficult. The method of the present invention simplifies that process and may provide key information for a better understanding of autism and other diseases in which the immune response of different individuals may provide an explanation for the differential development of disease in some individuals exposed to an agent or a group of agents, while others similarly exposed do not develop the disease.
Imbalances of the PIR, triggered by infection, may lead to many diseases, including cancers, leukemia, neuronal diseases (Alzheimer's, Multiple Sclerosis, Parkinson's, autism etc), autoimmune diseases, and metabolic diseases. These diseases may be called PIR diseases. There may be two PIR disease forms. (1) a “loss of function” form, and (2) a “gain of function” form. In the “loss of function” form, a person is susceptible to a disease because his/her restricted and/or limited PIR lacks the cells that produce the most efficient and necessary Igs and TRs. In the “gain of function” form, a person is susceptible to a disease because his/her PIR gained cells that produce Igs and TRs that normally should not be there. In the “loss of function” (LOF) PIR diseases, an individual does not have the appropriate functional B or T cells to fight a disease. His/her HLA typing determines that those cells are eliminated during the early stages of the immune cell maturation process, the cells generally being eliminated because they react to strongly to his/her own proteins.
One aspect of the invention also comprises entering a patient immune cell profile into a database in combination with identifying information such as, for example, a patient identification number, a code comprising the patient's HLA type, a disease code comprising one or more clinical diagnoses that may have been made, a “staging code” comprising the date of the sample, a cell type code comprising the type of cell subpopulation from which the RNA was amplified and sequenced, and one or more sequence codes comprising the sequences identified for the sample.
The described method includes a novel primer set that not only allows amplification of the entire immunorepertoire, but also allows multiplex amplification that is semi-quantitative. Multiplex amplification requires that only a few PCR or RT-PCR reactions are needed. For example, all immunoglobulin (Ig) sequences present may be amplified in one reaction, or two or three reactions may be performed separately, using primers specific for IgH, IgL or IgK. Similarly, the T-cell receptors (TRs) may be amplified in just one reaction, or may be amplified in a few reactions including the T-cell receptors designated TRA, TRB, TRD, and TRG. MHC genes may be amplified in just one PCR reaction. Semi-quantitative amplification allows all the targets in the multiplex reaction to be amplified independently, so that the end point analysis of the amplified products will reflect the original internal ratio among the targets. Because this ratio is maintained, it is possible to produce an immune cell profile that indicates the presence or absence, as well as relative numbers, of various immune system cells. Amplification of RNA according to the method of the invention may be performed using any or all of the primers listed in Tables 1, 2, and/or 3. The invention therefore provides a method for using at least one (which may, of course, more preferably include a least 2, at least 3, at least 4, etc.) primers chosen from among the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, and combinations thereof, to perform a first amplification producing at least one first amplicon, at least one of the target-specific primers containing additional base pairs so that the amplification results in the addition of a binding sequence for at least one common primer into the at least one first amplicon; amplifying the at least one first amplicon in a second amplification using at least one common primer to produce at least one second amplicon; and sequencing the at least one second amplicon to identify and quantify the sequences produced by the first and second amplifications. The listed primers were designed by the inventor to provide efficient amplification of their respective RNA and/or DNA targets. Use of the entire group of primers is effective to produce a detailed immune status profile for an individual. Use of a subset may, however, be desired when specific populations of T or B cells, for example, are the subject of particular interest.
By way of further explanation, the following example may be illustrative of the methods of the invention. Blood samples may be taken from children prior to administration of any vaccines, those blood samples for each child being used in the method of the invention to create a “baseline” immune status profile or personal immunorepertoire (PIR) from which future immune cell profiles, created from blood samples taken during later years and analyzed by the method of the invention, may be compared. For each child, the future samples may be utilized to determine whether there has been an exposure to an agent which has expanded a population of cells known to be correlated with a disease, and this may serve as a “marker” for the risk of development of the disease in the future. Individuals so identified may then be more closely monitored so that early detection is possible, and any available treatment options may be provided at an earlier stage in the disease process.
The method of the invention may be especially useful for identifying commonalities between individuals with autoimmune diseases, for example, and may provide epidemiological data that will better describe the correlation between infectious and environmental factors and diseases such as heart disease, atherosclerosis, diabetes, and cancer-providing biomarkers that signal either the presence of a disease, or the tendency to develop disease.
The method may also be useful for development of passive immunity therapies. For example, following exposure to an infectious agent, certain antibody-producing B cells and/or T cells are expanded. The method of the invention enables the identification of protective antibodies, for example, and those antibodies may be utilized to provide passive immunity therapies in situations where such therapy is needed.
The method of the invention may also provide the ability to accomplish targeted removal of cells with undesirable rearrangements, the method providing a means by which such cells rearrangements may be identified.
The inventor has identified and developed target-specific primers for use in the method of the invention. T-cell-specific primers are shown in Table 1, antibody-specific primers are shown in Table 2, and HLA-specific primers are shown in Table 3. Therefore, the method may comprise using any combination of primers of Table 1, Table 2, and/or Table 3 to amplify RNA and/or DNA from a blood sample, and more particularly to identify antibodies, T-cell receptors, and HLA molecules within a population of cells. For example, an analysis of T-cell distribution might utilize all or a portion of the primers listed in Table 1 (SEQ ID NO: 1 through SEQ ID NO: 157). An analysis of Ig might utilize all or a portion of the primers listed in Table 2 (SEQ ID NO: 158 through SEQ ID NO: 225), and an analysis of HLA distribution might utilize all or a portion of the primers listed in Table 3 (SEQ ID NO: 159 through SEQ ID NO: 312).
In a tem-PCR reaction, nested gene-specific primers are designed to enrich the targets during initial PCR cycling. Later, universal “Super” primers are used to amplify all targets. Primers are designated as Fo (forward out), Fi (forward in), Ri (reverse in), Ro (reverse out), FS (forward super primer) and RS, (reverse super primer), with super primers being common to a variety of the molecules due to the addition of a binding site for those primers at the end of a target-specific primer. The gene-specific primers (Fo, Fi, Ri, and Ro) are used at extremely low concentrations. Different primers are involved in the tem-PCR process at each of the three major stages. First, at the “enrichment” stage, low-concentration gene-specific primers are given enough time to find the templates. For each intended target, depending on which primers are used, four possible products may be generated: Fo/Ro, Fi/Ro, Fi/Ri, and Fo/Ri. The enrichment stage is typically carried out for 10 cycles. In the second, or “tagging” stage, the annealing temperature is raised to 72° C., and only the long 40-nucleotide inside primers (Fi and Ri) will work. After 10 cycles of this tagging stage, all PCR products are “tagged” with the universal super primer sequences. Then, at the third “amplification” stage, high-concentration super primers work efficiently to amplify all targets and label the pCR products with biotin during the process. Specific probes may be covalently linked with Luminex® color-coated beads.
To amplify the genes coding for immunoglobulin superfamily molecules, the inventor designed nested primers based on sequence information available in the public domain. For studying B and T cell VDJ rearrangement, the inventor designed primers to amplify rearranged and expressed RNAs. Generally, a pair of nested forward primers is designed from the V genes and a set of reverse nested primers are designed from the J or C genes. The average amplicon size is 250-350 bp. For the IgHV genes, for example, there are 123 genes that can be classified into 7 different families, and the present primers are designed to be family-specific. However, if sequencing the amplified cDNA sequences, there are enough sequence diversities to allow further differentiation among the genes within the same family. For the MHC gene locus, the intent is to amplify genomic DNA.
The invention may be further described by means of the following non-limiting examples.
EXAMPLES
Amplification of T or B Cell Rearrangement Sites
All oligos were resuspended using 1×TE. All oligos except 454A and 454B were resuspended to a concentration of 100 pmol/uL. 454A and 454B were resuspended to a concentration of 1000 pmol/uL 454A and 454B are functionally the same as the common primers described previously, the different sequences were used for follow up high throughput sequencing procedures.
Three different primer mixes were made. An Alpha Delta primer mix included 82 primers (all of TRAV-C+TRDV-C), a Beta Gamma primer mix included 79 primers (all of TRBVC and TRGV-C) and a B cell primer mix that included a total of 70 primers. Fo, Fi, and Ri primers were at a concentration of 1 pmol/μL. Ro primers were at a concentration of 5 pmol/uL. 454A and 454B were at a concentration of 30 pmol/μL.
Three different RNA samples were ordered from ALLCELLS (www.allcells.com). All samples were diluted down to a final concentration of 4 ng/uL. The samples used were: ALL-PB-MNC (from a patient with acute lymphoblastic leukemia), NPB-Pan T Cells (normal T cells) and NPB-B Cells (normal B cells).
RT-PCR was performed using a Qiagen® One-Step RT-PCR kit. Each sample contained the following:
10 μL of Qiagen® Buffer
2 μL of DNTP's
2 μl of Enzyme
23.5 μL of dH2O
10 μL of the appropriate primer mix
2.5 μL of the appropriate template (long of RNA total)
The samples were run using the following cycling conditions:
50° C. for 30 minutes
95° C. for 15 minutes
94° C. for 30 seconds
15 cycles of
55° C. for 1 minute
72° C. for 1 minute
94° C. for 15 seconds
6 cycles of
70° C. for 1 minute 30 seconds
94° C. for 15 seconds
30 cycles of
55° C. for 15 seconds
72° C. for 15 seconds
72° C. for 3 minutes
4° C. Hold
The order of samples placed in the gel shown in FIG. 1a was: (1) Ladder (500 bp being the largest working down in steps of 20 bp, the middle bright band in FIG. 1a is 200 bp); (2) α+δ primer mix with 10 ng Pan T Cells Template; (3) β+γ primer mix with 10 ng Pan T Cells Template; (4) B Cell primer mix with 10 ng B Cells Template; (5) B Cell primer mix with 10 ng ALL Cells Template; (6) α+δ primer mix with 10 ng ALL Cells Template; (7) β+γ primer mix with 10 ng ALL Cells Template; 8. α+δ primer mix blank; (9) β+γ primer mix blank; (10) B Cell primer mix blank; (11) Running buffer blank. These samples were run on a pre-cast ClearPAGE® SDS 10% gel using 1× ClearPAGE® DNA native running buffer.
The initial experiment showed that a smear is generated from PCR reactions where templates were included. The smears indicate different sizes of PCR products were generated that represented a mixture of different VDJ rearrangements. There was some background amplification from the B cell reaction. Further improvement on that primer mix cleaned up the reaction.
To determine whether the PCR products indeed include different VDJ rearrangements, it was necessary to isolate and sequence the single clones. Instead of using the routine cloning procedures, the inventor used a different strategy. PCR products generated from the Alpha Delta mix and the Beta Gamma mix (lanes 2 and 3 in FIG. 1a) were diluted 1:1000 and a 2 μl aliquot used as PCR template in the following reaction. Then, instead of using a mixture of primers that targeting the entire repertoire, one pair of specific Fi and Ri primers were used (5 pmol each) to amplify only one specific PCR product. The following cycling conditions were used to amplify the samples:
95° C. for 5 minutes
30 cycles of
94° C. for 30 seconds
72° C. for 1 minute
72° C. for 3 minutes
4° C. hold
A Qiagen PCR kit was used to amplify the products. The Master Mix used for the PCR contained the following: 5 μL 10×PCR Buffer, 1 μL dNTP, 0.25 μL HotStartTaq Plus, and 39.75 μL H2O. (For a mix for 12 reactions: 60 μL 10×PCR Buffer, 12 μL dNTP, 3 μL HotStartTaq Plus, and 477 μL H2O.)
The photograph of the gel in FIG. 1b shows the PCR products of the following reactions: (1) Ladder; (2) TRAV1Fi+TRACRi with alpha delta Pan T PCR product; (3) TRAV2Fi+TRACRi with alpha delta Pan T PCR product; (4) TRAV3Fi+TRACRi with alpha delta Pan T PCR product; (5) TRAV4Fi+TRACRi with alpha delta Pan T PCR product; (6) TRAV5Fi+TRACRi with alpha delta Pan T PCR product; (7) TRAV1Fi+TRACRi with alpha delta Pan T PCR product; (8) TRAV2Fi+TRACRi with alpha delta Pan T PCR product; (9) TRAV3Fi+TRACRi with alpha delta Pan T PCR product; (10) TRAV4Fi+TRACRi with alpha delta Pan T PCR product; (11) TRAV5Fi+TRACRi with alpha delta Pan T PCR product; (12) PCR Blank. Primers listed as F1 are “forward inner” primers and primers listed as Fo are “forward outer” primers, with Ri and Ro indicating “reverse inner” and “reverse outer” primers, respectively.
As illustrated by FIG. 1b, a single PCR product was generated from each reaction. Different size bands were generated from different reactions. This PCR cloning approach is successful for two major reasons—(1) The PCR templates used in this reaction were diluted PCR products (1:1000) of previous reactions that used primer mixes to amplify all possible VDJ rearrangements (for example, a primer mix was used that included total of 82 primers to amplify T cell receptor Alpha and Delta genes) and (2) Only one pair of PCR primers, targeting a specific V gene, are used in each reaction during this “cloning” experiment. In every case, a single clone was obtained, and a specific T cell receptor V gene that matched the Fi primer was identified.
Sequencing of Immune Cell RNA Using Primers of SEQ ID NO: 1-SEQ ID NO: 312
Pan-T, pan-B, and neutrophil isolation was performed using super-paramagnetic polystyrene beads coated with monoclonal antibody specific for certain cell types (Dynabeads®, Invitrogen Corp., Carlsbad, Calif.) following manufacturer's instructions. Anti-CD3 beads were used to isolate pan-T cells, anti-CD19 beads for pan-B cells, and anti-CD15 beads for neutrophils. Isolated cells were resuspended in 300 μl RNAProtect® (Qiagen) reagent and counted using a hemacytometer.
T cell subpopulations were isolated from a normal patient 48-year-old Asian male. PMBCs were obtained from 40 ml of whole blood collected in sodium heparin by density centrifugation over Ficoll Prep Plus Reagent. Pan-T cells were isolated from the mononuclear layer using a magnetic bead isolation kit (Miltenyi Biotec, Auburn, Calif.), following manufacturer's instructions. Anti-CD4 and anti-CD25 beads were used to isolate regulatory T cells, anti-CD56 for NKT cells, anti-CD8 for cytotoxic T cells, and anti-CD4 and anti-CD294 for Th2 cells. Th1 cells were isolated via negative selection. A separate 40 ml sample of whole blood collected in sodium heparin was used to obtain naive, activated, and memory T cell subpopulations. Anti-CD45RA beads were used to isolate naive T cells, anti-CD69 for activated T cells and anti-CD45RO beads for memory T cells. Isolated cells were re-suspended in 300 μl of RNAProtect® reagent (Qiagen). Cells were counted using a hemacytometer.
DNA extraction from the isolated neutrophils was performed using a QIAmp® DNA mini kit (Qiagen) using the protocol provided by the manufacturer. RNA was extracted from T cell subsets using an Rneasy® kit (Qiagen) according to the protocol provided by the manufacturer. The concentrations of extracted DNA and RNA were measured using Nanodrop® technology (Nanodrop Technologies, Wilmington, Del.). Samples were stored at −80° C.
RT-PCR was performed according to the method of the invention using nested PCR to amplify multiple targets and target-specific primers to incorporate a common primer binding sequence into the resulting amplicons in a first amplification reaction. Common primers were then used in a second amplification reaction to exponentially amplify the amplicons rescued from the first amplification reaction while preserving the relative ratios of each amplicon. PCR was performed using a One-Step RT-PCR kit (Qiagen). DNA amplification for HLA typing was similarly performed, but with a mulitplex PCR kit (Qiagen). Each amplicon mixture was subjected to high-throughput sequencing with the Roche 454 sequencing platform.
More than 1.6 million effective sequences were generated for one single individual (normal 48-year-old Asian male) by sampling different subpopulations of lymphocytes in peripheral blood at different time points. Additionally, 170,734 effective sequences were generated for the colon cancer, CLL, SLE, and a second healthy patient (a 32-year-old Caucasian male). The number of unique reads generated in this study was compared to the number of unique reads existing in public databases in Table 1. The public sequence data set was compiled by searching Genbank nucleotide database with terms of ‘human[orgn] AND (immunoglobulin[titl] OR T-cell receptor[titl]) AND mRNA[titl]’. In addition, the annotated IMGT/LIGM-DB (Brezinschek et al, 1995) cDNA sequences were gathered with a Python script. The two data sets were merged, and one copy was kept for any redundant sequences.
Biased usage of V, and J gene segments in a healthy control, CLL, colon cancer, and SLE sample was analyzed. The bias of domain usage was particularly outstanding for TCR beta chain in the colon cancer sample and SLE sample, while in the healthy control sample the domain usage is quite normal without significant bias to any particular domain. It was evident that colon cancer and SLE profiles not only show clonal expansion, but demonstrate the loss of overall diversity, as well.
The distribution of functional germline V, J gene segments seen in the pan-T and pan-B populations from normal patient indicated that 87.2% of potential combinations have sequences observed. Only IGHV3-d was not observed in this investigation, while TRBV4-3, IGHV3-d, IGHV4-30-4 and IGHV4-31 and IGHL3-22 were observed in other samples with extremely low frequency. Previous research did not reveal any cDNA sequence data related to IGHV3-d, which suggests that IGHV3-d may be used infrequently. Some sequences were present in high (e.g. 1000) numbers, while others were present in significantly lower numbers. The inventor believes that higher numbers represent lymphocyte clonal expansions, reflecting the real immune responses in the subject. Studies of VH gene distribution in normal individuals have previously found the frequency of usage in general to be similar to the germline complexity, while many immune responses show some level of bias in the usage of V, D and J gene segments.









TABLE 1







Sequence


Primer
Sequence
ID Number







TRAV1Fo
5′-TGCACGTACCAGACATCTGG-3′
SEQ ID NO: 1


 


TRAV1Fi
AGGTCGTTTTTCTTCATTCC
SEQ ID NO: 2


 


TRAV2Fo
TCTGTAATCACTCTGTGTCC
SEQ ID NO: 3


 


TRAV2Fi
AGGGACGATACAACATGACC
SEQ ID NO: 4


 


TRAV3Fo
CTATTCAGTCTCTGGAAACC
SEQ ID NO: 5


 


TRAV3Fi
ATACATCACAGGGGATAACC
SEQ ID NO: 6


 


TRAV4Fo
TGTAGCCACAACAACATTGC
SEQ ID NO: 7


 


TRAV4Fi
AAAGTTACAAACGAAGTGGC
SEQ ID NO: 8


 


TRAV5Fo
GCACTTACACAGACAGCTCC
SEQ ID NO: 9


 


TRAV5Fi
TATGGACATGAAACAAGACC
SEQ ID NO: 10


 


TRAV6Fo
GCAACTATACAAACTATTCC
SEQ ID NO: 11


 


TRAV6Fi
GTTTTCTTGCTACTCATACG
SEQ ID NO: 12


 


TRAV7Fo
TGCACGTACTCTGTCAGTCG
SEQ ID NO: 13


 


TRAV7Fi
GGATATGAGAAGCAGAAAGG
SEQ ID NO: 14


 


TRAV8Fo
AATCTCTTCTGGTATGTSCA
SEQ ID NO: 15


 


TRAV8Fi
GGYTTTGAGGCTGAATTTA
SEQ ID NO: 16


 


TRAV9Fo
GTCCAATATCCTGGAGAAGG
SEQ ID NO: 17


 


TRAV9Fi
AACCACTTCTTTCCACTTGG
SEQ ID NO: 18


 


TRAV10Fo
AATGCAATTATACAGTGAGC
SEQ ID NO: 19


 


TRAV10Fi
TGAGAACACAAAGTCGAACG
SEQ ID NO: 20


 


TRAV11Fo
TCTTAATTGTACTTATCAGG
SEQ ID NO: 21


 


TRAV11Fi
TCAATCAAGCCAGAAGGAGC
SEQ ID NO: 22


 


TRAV12Fo
TCAGTGTTCCAGAGGGAGCC
SEQ ID NO: 23


 


TRAV12Fi
ATGGAAGGTTTACAGCACAG
SEQ ID NO: 24


 


TRAV13Fo
ACCCTGAGTGTCCAGGAGGG
SEQ ID NO: 25


 


TRAV13Fi
TTATAGACATTCGTTCAAAT
SEQ ID NO: 26


 


TRAV14Fo
TGGACTGCACATATGACACC
SEQ ID NO: 27


 


TRAV14Fi
CAGCAAAATGCAACAGAAGG
SEQ ID NO: 28


 


TRAV16Fo
AGCTGAAGTGCAACTATTCC
SEQ ID NO: 29


 


TRAV16Fi
TCTAGAGAGAGCATCAAAGG
SEQ ID NO: 30


 


TRAV17Fo
AATGCCACCATGAACTGCAG
SEQ ID NO: 31


 


TRAV17Fi
GAAAGAGAGAAACACAGTGG
SEQ ID NO: 32


 


TRAV18Fo
GCTCTGACATTAAACTGCAC
SEQ ID NO: 33


 


TRAV18Fi
CAGGAGACGGACAGCAGAGG
SEQ ID NO: 34


 


TRAV19Fo
ATGTGACCTTGGACTGTGTG
SEQ ID NO: 35


 


TRAV19Fi
GAGCAAAATGAAATAAGTGG
SEQ ID NO: 36


 


TRAV20Fo
ACTGCAGTTACACAGTCAGC
SEQ ID NO: 37


 


TRAV20Fi
AGAAAGAAAGGCTAAAAGCC
SEQ ID NO: 38


 


TRAV21Fo
ACTGCAGTTTCACTGATAGC
SEQ ID NO: 39


 


TRAV21Fi
CAAGTGGAAGACTTAATGCC
SEQ ID NO: 40


 


TRAV22Fo
GGGAGCCAATTCCACGCTGC
SEQ ID NO: 41


 


TRAV22Fi
ATGGAAGATTAAGCGCCACG
SEQ ID NO: 42


 


TRAV23Fo
ATTTCAATTATAAACTGTGC
SEQ ID NO: 43


 


TRAV23Fi
AAGGAAGATTCACAATCTCC
SEQ ID NO: 44


 


TRAV24Fo
GCACCAATTTCACCTGCAGC
SEQ ID NO: 45


 


TRAV24Fi
AGGACGAATAAGTGCCACTC
SEQ ID NO: 46


 


TRAV25Fo
TCACCACGTACTGCAATTCC
SEQ ID NO: 47


 


TRAV25Fi
AGACTGACATTTCAGTTTGG
SEQ ID NO: 48


 


TRAV26Fo
TCGACAGATTCMCTCCCAGG
SEQ ID NO: 49


 


TRAV26Fi
GTCCAGYACCTTGATCCTGC
SEQ ID NO: 50


 


TRAV27Fo
CCTCAAGTGTTTTTTCCAGC
SEQ ID NO: 51


 


TRAV27Fi
GTGACAGTAGTTACGGGTGG
SEQ ID NO: 52


 


TRAV29Fo
CAGCATGTTTGATTATTTCC
SEQ ID NO: 53


 


TRAV29Fi
ATCTATAAGTTCCATTAAGG
SEQ ID NO: 54


 


TRAV30Fo
CTCCAAGGCTTTATATTCTG
SEQ ID NO: 55


 


TRAV30Fi
ATGATATTACTGAAGGGTGG
SEQ ID NO: 56


 


TRAV34Fo
ACTGCACGTCATCAAAGACG
SEQ ID NO: 57


 


TRAV34Fi
TTGATGATGCTACAGAAAGG
SEQ ID NO: 58


 


TRAV35Fo
TGAACTGCACTTCTTCAAGC
SEQ ID NO: 59


 


TRAV35Fi
CTTGATAGCCTTATATAAGG
SEQ ID NO: 60


 


TRAV36Fo
TCAATTGCAGTTATGAAGTG
SEQ ID NO: 61


 


TRAV36Fi
TTTATGCTAACTTCAAGTGG
SEQ ID NO: 62


 


TRAV38Fo
GCACATATGACACCAGTGAG
SEQ ID NO: 63


 


TRAV38Fi
TCGCCAAGAAGCTTATAAGC
SEQ ID NO: 64


 


TRAV39Fo
TCTACTGCAATTATTCAACC
SEQ ID NO: 65


 


TRAV39Fi
CAGGAGGGACGATTAATGGC
SEQ ID NO: 66


 


TRAV40Fo
TGAACTGCACATACACATCC
SEQ ID NO: 67


 


TRAV40Fi
ACAGCAAAAACTTCGGAGGC
SEQ ID NO: 68


 


TRAV41Fo
AACTGCAGTTACTCGGTAGG
SEQ ID NO: 69


 


TRAV41Fi
AAGCATGGAAGATTAATTGC
SEQ ID NO: 70


 


TRACRo
GCAGACAGACTTGTCACTGG
SEQ ID NO: 71


 


TRACRi
AGTCTCTCAGCTGGTACACG
SEQ ID NO: 72


 


TRBV1Fo
AATGAAACGTGAGCATCTGG
SEQ ID NO: 73


 


TRBV1Fi
CATTGAAAACAAGACTGTGC
SEQ ID NO: 74


 


TRBV2Fo
GTGTCCCCATCTCTAATCAC
SEQ ID NO: 75


 


TRBV2Fi
TGAAATCTCAGAGAAGTCTG
SEQ ID NO: 76


 


TRBV3Fo
TATGTATTGGTATAAACAGG
SEQ ID NO: 77


 


TRBV3Fi
CTCTAAGAAATTTCTGAAGA
SEQ ID NO: 78


 


TRBV4Fo
GTCTTTGAAATGTGAACAAC
SEQ ID NO: 79


 


TRBV4Fi
GGAGCTCATGTTTGTCTACA
SEQ ID NO: 80


 


TRBV5Fo
GATCAAAACGAGAGGACAGC
SEQ ID NO: 81


 


TRBV5aFi
CAGGGGCCCCAGTTTATCTT
SEQ ID NO: 82


 


TRBV5bFi
GAAACARAGGAAACTTCCCT
SEQ ID NO: 83


 


TRBV6aFo
GTGTGCCCAGGATATGAACC
SEQ ID NO: 84


 


TRBV6bFo
CAGGATATGAGACATAATGC
SEQ ID NO: 85


 


TRBV6aFi
GGTATCGACAAGACCCAGGC
SEQ ID NO: 86


 


TRBV6bFi
TAGACAAGATCTAGGACTGG
SEQ ID NO: 87


 


TRBV7Fo
CTCAGGTGTGATCCAATTTC
SEQ ID NO: 88


 


TRBV7aFi
TCTAATTTACTTCCAAGGCA
SEQ ID NO: 89


 


TRBV7bFi
TCCCAGAGTGATGCTCAACG
SEQ ID NO: 90


 


TRBV7cFi
ACTTACTTCAATTATGAAGC
SEQ ID NO: 91


 


TRBV7dFi
CCAGAATGAAGCTCAACTAG
SEQ ID NO: 92


 


TRBV9Fo
GAGACCTCTCTGTGTACTGG
SEQ ID NO: 93


 


TRBV9Fi
CTCATTCAGTATTATAATGG
SEQ ID NO: 94


 


TRBV10Fo
GGAATCACCCAGAGCCCAAG
SEQ ID NO: 95


 


TRBV10Fi
GACATGGGCTGAGGCTGATC
SEQ ID NO: 96


 


TRBV11Fo
CCTAAGGATCGATTTTCTGC
SEQ ID NO: 97


 


TRBV11Fi
ACTCTCAAGATCCAGCCTGC
SEQ ID NO: 98


 


TRBV12Fo
AGGTGACAGAGATGGGACAA
SEQ ID NO: 99


 


TRBV12aFi
TGCAGGGACTGGAATTGCTG
SEQ ID NO: 100


 


TRBV12bFi
GTACAGACAGACCATGATGC
SEQ ID NO: 101


 


TRBV13Fo
CTATCCTATCCCTAGACACG
SEQ ID NO: 102


 


TRBV13Fi
AAGATGCAGAGCGATAAAGG
SEQ ID NO: 103


 


TRBV14Fo
AGATGTGACCCAATTTCTGG
SEQ ID NO: 104


 


TRBV14Fi
AGTCTAAACAGGATGAGTCC
SEQ ID NO: 105


 


TRBV15Fo
TCAGACTTTGAACCATAACG
SEQ ID NO: 106


 


TRBV15Fi
AAAGATTTTAACAATGAAGC
SEQ ID NO: 107


 


TRBV16Fo
TATTGTGCCCCAATAAAAGG
SEQ ID NO: 108


 


TRBV16Fi
AATGTCTTTGATGAAACAGG
SEQ ID NO: 109


 


TRBV17Fo
ATCCATCTTCTGGTCACATG
SEQ ID NO: 110


 


TRBV17Fi
AACATTGCAGTTGATTCAGG
SEQ ID NO: 111


 


TRBV18Fo
GCAGCCCAATGAAAGGACAC
SEQ ID NO: 112


 


TRBV18Fi
AATATCATAGATGAGTCAGG
SEQ ID NO: 113


 


TRBV19Fo
TGAACAGAATTTGAACCACG
SEQ ID NO: 114


 


TRBV19Fi
TTTCAGAAAGGAGATATAGC
SEQ ID NO: 115


 


TRBV20Fo
TCGAGTGCCGTTCCCTGGAC
SEQ ID NO: 116


 


TRBV20Fi
GATGGCAACTTCCAATGAGG
SEQ ID NO: 117


 


TRBV21Fo
GCAAAGATGGATTGTGTTCC
SEQ ID NO: 118


 


TRBV21Fi
CGCTGGAAGAAGAGCTCAAG
SEQ ID NO: 119


 


TRBV23Fo
CATTTGGTCAAAGGAAAAGG
SEQ ID NO: 120


 


TRBV23Fi
GAATGAACAAGTTCTTCAAG
SEQ ID NO: 121


 


TRBV24Fo
ATGCTGGAATGTTCTCAGAC
SEQ ID NO: 122


 


TRBV24Fi
GTCAAAGATATAAACAAAGG
SEQ ID NO: 123


 


TRBV25Fo
CTCTGGAATGTTCTCAAACC
SEQ ID NO: 124


 


TRBV25Fi
TAATTCCACAGAGAAGGGAG
SEQ ID NO: 125


 


TRBV26Fo
CCCAGAATATGAATCATGTT
SEQ ID NO: 126


 


TRBV26Fi
ATTCACCTGGCACTGGGAGC
SEQ ID NO: 127


 


TRBV27Fo
TTGTTCTCAGAATATGAACC
SEQ ID NO: 128


 


TRBV27Fi
TGAGGTGACTGATAAGGGAG
SEQ ID NO: 129


 


TRBV28Fo
ATGTGTCCAGGATATGGACC
SEQ ID NO: 130


 


TRBV28Fi
AAAAGGAGATATTCCTGAGG
SEQ ID NO: 131


 


TRBV29Fo
TCACCATGATGTTCTGGTAC
SEQ ID NO: 132


 


TRBV29Fi
CTGGACAGAGCCTGACACTG
SEQ ID NO: 133


 


TRBV30Fo
TGTGGAGGGAACATCAAACC
SEQ ID NO: 134


 


TRBV30Fi
TTCTACTCCGTTGGTATTGG
SEQ ID NO: 135


 


TRBCRo
GTGTGGCCTTTTGGGTGTGG
SEQ ID NO: 136


 


TRBCRi
TCTGATGGCTCAAACACAGC
SEQ ID NO: 137


 


TRDV1Fo
TGTATGAAACAAGTTGGTGG
SEQ ID NO: 138


 


TRDV1Fi
CAGAATGCAAAAAGTGGTCG
SEQ ID NO: 139


 


TRDV2Fo
ATGAAAGGAGAAGCGATCGG
SEQ ID NO: 140


 


TRDV2Fi
TGGTTTCAAAGACAATTTCC
SEQ ID NO: 141


 


TRDV3Fo
GACACTGTATATTCAAATCC
SEQ ID NO: 142


 


TRDV3Fi
GCAGATTTTACTCAAGGACG
SEQ ID NO: 143


 


TRDCRo
AGACAAGCGACATTTGTTCC
SEQ ID NO: 144


 


TRDCRi
ACGGATGGTTTGGTATGAGG
SEQ ID NO: 145


 


TRGV1-5Fo
GGGTCATCTGCTGAAATCAC
SEQ ID NO: 146


 


TRGV1-
AGGAGGGGAAGGCCCCACAG
SEQ ID NO: 147


5, 8Fi


 


TRGV8Fo
GGGTCATCAGCTGTAATCAC
SEQ ID NO: 148


 


TRGV5pFi
AGGAGGGGAAGACCCCACAG
SEQ ID NO: 149


 


TRGV9Fo
AGCCCGCCTGGAATGTGTGG
SEQ ID NO: 150


 


TRGV9Fi
GCACTGTCAGAAAGGAATCC
SEQ ID NO: 151


 


TRGV10Fo
AAGAAAAGTATTGACATACC
SEQ ID NO: 152


 


TRGV10Fi
ATATTGTCTCAACAAAATCC
SEQ ID NO: 153


 


TRGV11Fo
AGAGTGCCCACATATCTTGG
SEQ ID NO: 154


 


TRGV11Fi
GCTCAAGATTGCTCAGGTGG
SEQ ID NO: 155


 


TRGCRo
GGATCCCAGAATCGTGTTGC
SEQ ID NO: 156


 


TRGCRi
GGTATGTTCCAGCCTTCTGG
SEQ ID NO: 157


















TABLE 2







Sequence


Primer
Sequence
ID Number







IgHV1aFo
AGTGAAGGTCTCCTGCAAGG
SEQ ID NO: 158


 


IgHV1bFo
AGTGAAGGTTTCCTGCAAGG
SEQ ID NO: 159


 


IgHV1aFi
AGTTCCAGGGCAGAGTCAC
SEQ ID NO: 160


 


IgHV1bFi
AGTTTCAGGGCAGGGTCAC
SEQ ID NO: 161


 


IgHV1cFi
AGTTCCAGGAAAGAGTCAC
SEQ ID NO: 162


 


IgHV1dFi
AATTCCAGGACAGAGTCAC
SEQ ID NO: 163


 


IgHV2Fo
TCTCTGGGTTCTCACTCAGC
SEQ ID NO: 164


 


IgHV2Fi
AAGGCCCTGGAGTGGCTTGC
SEQ ID NO: 165


 


IgHV3aFo
TCCCTGAGACTCTCCTGTGC
SEQ ID NO: 166


 


IgHV3bFo
CTCTCCTGTGCAGCCTCTGG
SEQ ID NO: 167


 


IgHV3cFo
GGTCCCTGAGACTCTCCTGT
SEQ ID NO: 168


 


IgHV3dFo
CTGAGACTCTCCTGTGTAGC
SEQ ID NO: 169


 


IgHV3aFi
CTCCAGGGAAGGGGCTGG
SEQ ID NO: 170


 


IgHV3bFi
GGCTCCAGGCAAGGGGCT
SEQ ID NO: 171


 


IgHV3cFi
ACTGGGTCCGCCAGGCTCC
SEQ ID NO: 172


 


IgHV3dFi
GAAGGGGCTGGAGTGGGT
SEQ ID NO: 173


 


IgHV3eFi
AAAAGGTCTGGAGTGGGT
SEQ ID NO: 174


 


IgHV4Fo
AGACCCTGTCCCTCACCTGC
SEQ ID NO: 175


 


IgHV4Fi
AGGGVCTGGAGTGGATTGGG
SEQ ID NO: 176


 


IgHV5Fo
GCGCCAGATGCCCGGGAAAG
SEQ ID NO: 177


 


IgHV5Fi
GGCCASGTCACCATCTCAGC
SEQ ID NO: 178


 


IgHV6Fo
CCGGGGACAGTGTCTCTAGC
SEQ ID NO: 179


 


IgHV6Fi
GCCTTGAGTGGCTGGGAAGG
SEQ ID NO: 180


 


IgHV7Fo
GTTTCCTGCAAGGCTTCTGG
SEQ ID NO: 181


 


IgHV7Fi
GGCTTGAGTGGATGGGATGG
SEQ ID NO: 182


 


IgHJRo
ACCTGAGGAGACGGTGACC
SEQ ID NO: 183


 


IgHJ1Ri
CAGTGCTGGAAGTATTCAGC
SEQ ID NO: 184


 


IgHJ2Ri
AGAGATCGAAGTACCAGTAG
SEQ ID NO: 185


 


IgHJ3Ri
CCCCAGATATCAAAAGCATC
SEQ ID NO: 186


 


IgHJ4Ri
GGCCCCAGTAGTCAAAGTAG
SEQ ID NO: 187


 


IgHJ5Ri
CCCAGGGGTCGAACCAGTTG
SEQ ID NO: 188


 


IgHJ6Ri
CCCAGACGTCCATGTAGTAG
SEQ ID NO: 189


 


IgKV1Fo
TAGGAGACAGAGTCACCATC
SEQ ID NO: 190


 


IgKV1Fi
TTCAGYGRCAGTGGATCTGG
SEQ ID NO: 191


 


IgKV2Fo
GGAGAGCCGGCCTCCATCTC
SEQ ID NO: 192


 


IgKV2aFi
TGGTACCTGCAGAAGCCAGG
SEQ ID NO: 193


 


IgKV2bFi
CTTCAGCAGAGGCCAGGCCA
SEQ ID NO: 194


 


IgKV3-7Fo
GCCTGGTACCAGCAGAAACC
SEQ ID NO: 195


 


IgKV3Fi
GCCAGGTTCAGTGGCAGTGG
SEQ ID NO: 196


 


IgKV6-7Fi
TCGAGGTTCAGTGGCAGTGG
SEQ ID NO: 197


 


IgKV4-5Fi
GACCGATTCAGTGGCAGCGG
SEQ ID NO: 198


 


IgKCRo
TTCAACTGCTCATCAGATGG
SEQ ID NO: 199


 


IgKCRi
ATGAAGACAGATGGTGCAGC
SEQ ID NO: 200


 


IgLV1aFo
GGGCAGAGGGTCACCATCTC
SEQ ID NO: 201


 


IgLV1bFo
GGACAGAAGGTCACCATCTC
SEQ ID NO: 202


 


IgLV1aFi
TGGTACCAGCAGCTCCCAGG
SEQ ID NO: 203


 


IgLV1bFi
TGGTACCAGCAGCTTCCAGG
SEQ ID NO: 204


 


IgLV2Fo
CTGCACTGGAACCAGCAGTG
SEQ ID NO: 205


 


IgLV2Fi
TCTCTGGCTCCAAGTCTGGC
SEQ ID NO: 206


 


IgLV3aFo
ACCAGCAGAAGCCAGGCCAG
SEQ ID NO: 207


 


IgLV3bFo
GAAGCCAGGACAGGCCCCTG
SEQ ID NO: 208


 


IgLV3aFi
CTGAGCGATTCTCTGGCTCC
SEQ ID NO: 209


 


IgLV3bFi
TTCTCTGGGTCCACCTCAGG
SEQ ID NO: 210


 


IgLV3cFi
TTCTCTGGCTCCAGCTCAGG
SEQ ID NO: 211


 


IgLV4Fo
TCGGTCAAGCTCACCTGCAC
SEQ ID NO: 212


 


IgLV4Fi
GGGCTGACCGCTACCTCACC
SEQ ID NO: 213


 


IgLV5Fo
CAGCCTGTGCTGACTCAGCC
SEQ ID NO: 214


 


IgLV5Fi
CCAGCCGCTTCTCTGGATCC
SEQ ID NO: 215


 


IgLV6Fo
CCATCTCCTGCACCCGCAGC
SEQ ID NO: 216


 


IgLV7-8Fo
TCCCCWGGAGGGACAGTCAC
SEQ ID NO: 217


 


IgLV9,
CTCMCCTGCACCCTGAGCAG
SEQ ID NO: 218


11Fo


 


IgLV10Fo
AGACCGCCACACTCACCTGC
SEQ ID NO: 219


 


IgLV6, 8Fi
CTGATCGSTTCTCTGGCTCC
SEQ ID NO: 220


 


IgLV7Fi
CTGCCCGGTTCTCAGGCTCC
SEQ ID NO: 221


 


IgLV9Fi
ATCCAGGAAGAGGATGAGAG
SEQ ID NO: 222


 


IgLV10-
CTCCAGCCTGAGGACGAGGC
SEQ ID NO: 223


11Fi


 


IgLC1-7Ro
GCTCCCGGGTAGAAGTCACT
SEQ ID NO: 224


 


IgLC1-7Ri
AGTGTGGCCTTGTTGGCTTG
SEQ ID NO: 225


















TABLE 3





Primer
Sequence
Sequence ID Number







HLAI
CCCACTCCATGAGGTATTTC
SEQ ID NO: 226


Fo11


 


HLAI
CCTACTCCATGAGGTATTTC
SEQ ID NO: 227


Fo12


 


HLAI
GCGGGGAGCCCCGCTTCATC
SEQ ID NO: 228


Fo31


 


HLAI
GCGGGAAGCCCCGCTTCATC
SEQ ID NO: 229


Fo32


 


HLAI
GTGGAGAGCCCCGCTTCATC
SEQ ID NO: 230


Fo33


 


HLAI
GCGGAAAGCCCCGCTTCATC
SEQ ID NO: 231


Fo34


 


HLAI
GCGGAAAGCCCCACTTCATC
SEQ ID NO: 232


Fo35


 


HLAI
GCGGGAAGCCCCACTTCATC
SEQ ID NO: 233


Fo36


 


HLAI
GTGGGCTACGTGGACGACAC
SEQ ID NO: 234


Fi11


 


HLAI
GTGGGCTACGTGGACGGCAC
SEQ ID NO: 235


Fi12


 


HLAI-
GTTCGTGCGGTTCGACAGCG
SEQ ID NO: 236


Fi21


 


HLAI-
GTTCGTGCGGTTTGACAGCG
SEQ ID NO: 237


Fi22


 


HLAI-
GTTCGTGAGGTTCGACAGCG
SEQ ID NO: 238


Fi23


 


HLAI
TAATCCTTGCCGTCGTAGGC
SEQ ID NO: 239


Ri11


 


HLAI
TAATCCTTGCCGTCGTAAGC
SEQ ID NO: 240


Ri12


 


HLAI
TAATCTTTGCCGTCGTAGGC
SEQ ID NO: 241


Ri13


 


HLAI
GGTCCTCGTTCAGGGCGATG
SEQ ID NO: 242


Ro11


 


HLAI
GGTCCTCTTTCAGGGCGATG
SEQ ID NO: 243


Ro12


 


HLAI
GGTCCTCGTTCAAGGCGATG
SEQ ID NO: 244


Ro13


 


HLAI
GATCCTCGTTCAGGGCGATG
SEQ ID NO: 245


Ro14


 


HLAI
GGTCCTCATTCAGGGCGATG
SEQ ID NO: 246


Ro15


 


HLAI-
GCCTACGACGGCAAGGATTA
SEQ ID NO: 247


Fo41


 


HLAI-
GCTTACGACGGCAAGGATTA
SEQ ID NO: 248


Fo42


 


HLAI-
GCCTACGACGGCAAAGATTA
SEQ ID NO: 249


Fo43


 


HLAI-
CATCGCCCTGAACGAGGACC
SEQ ID NO: 250


Fi31


 


HLAI-
CATCGCCCTGAAAGAGGACC
SEQ ID NO: 251


Fi32


 


HLAI-
CATCGCCTTGAACGAGGACC
SEQ ID NO: 252


Fi33


 


HLAI-
CATCGCCCTGAACGAGGATC
SEQ ID NO: 253


Fi34


 


HLAI-
CATCGCCCTGAATGAGGACC
SEQ ID NO: 254


Fi35


 


HLAI-
GGTATCTGCGGAGCCCGTCC
SEQ ID NO: 255


Ri21


 


HLAI-
GGTATCTGCGGAGCCACTCC
SEQ ID NO: 256


Ri22


 


HLAI-
GGTGTCTGCGGAGCCACTCC
SEQ ID NO: 257


Ri23


 


HLAI-
GGTATCCGCGGAGCCACTCC
SEQ ID NO: 258


Ri24


 


HLAI-
GCAGCGTCTCCTTCCCGTTC
SEQ ID NO: 259


Ro21


 


HLAI-
CCAGCTTGTCCTTCCCGTTC
SEQ ID NO: 260


Ro22


 


HLAI-
CCAGCGTGTCCTTCCCGTTC
SEQ ID NO: 261


Ro23


 


HLAI-
GCAGCGTCTCCTTCCCATTC
SEQ ID NO: 262


Ro24


 


HLAI-
GCAGCGTCTCCTTCCKGTTC
SEQ ID NO: 263


Ro25


 


DRB17
TGTCATTTCTTCAATGGGAC
SEQ ID NO: 264


Fo11


 


DRB1
AGTGTCATTTCTTCAACGGG
SEQ ID NO: 265


Fo12


 


DRB1
GTGTTATTTCTTCAATGGGA
SEQ ID NO: 266


Fo13


 


DRB1
GTGTCAATTCTTCAATGGGA
SEQ ID NO: 267


Fo14


 


DRB4 Fo
GTGTCATTTCCTCAATGGGA
SEQ ID NO: 268


 


DRB1
GGAGCGGGTGCGGTTGCTGG
SEQ ID NO: 269


Fi11


 


DRB1
GGAGCGGGTGCGGTACCTGG
SEQ ID NO: 270


Fi12


 


DRB1
GGAGCGGGTGCGGTTCCTGG
SEQ ID NO: 271


Fi13


 


DRB1
GGAGCGGGTGCGATTCCTGG
SEQ ID NO: 272


Fi14


 


DRB1
GGAGCGGGTGCGGTATCTGC
SEQ ID NO: 273


Fi15


 


DRB1
GGAGCGGGTGCGGTTACTGG
SEQ ID NO: 274


Fi16


 


DRB45 Fi
ACATCTATAACCAAGAGGAG
SEQ ID NO: 275


 


DRB6 Fi
ACATCCATAAACGGGAGGAG
SEQ ID NO: 276


 


DRB7 Fi
TATAACCAAGAGGAGTACGT
SEQ ID NO: 277


 


DRB Ri11
AACCCCGTAGTTGTGTCTGC
SEQ ID NO: 278


 


DRB4 Ri
AACCCCGTAGTTGTATCTGC
SEQ ID NO: 279


 


DRB6 Ri
CGTAATTGTATCTGCAGTAG
SEQ ID NO: 280


 


DRB7 Ri
TAGTTGTCCACTTCGGCCCG
SEQ ID NO: 281


 


DRB Ro1
CGCTGCACTGTGAAGCTCTC
SEQ ID NO: 282


 


DRB Ro2
CGCTGCACCGTGAAGCTCTC
SEQ ID NO: 283


 


DRA Fo
CCTGTGGAACTGAGAGAGCC
SEQ ID NO: 284


 


DRA Fi
CAACGTCCTCATCTGTTTCA
SEQ ID NO: 285


 


DRA Ri
CTGCTGCATTGCTTTTGCGC
SEQ ID NO: 286


 


DRA Ro
TTACAGAGGCCCCCTGCGTT
SEQ ID NO: 287


 


DPB Fo
GTCCAGGGCAGGGCCACTCC
SEQ ID NO: 288


 


DPB Fi11
AATTACGTGTACCAGGGACG
SEQ ID NO: 289


 


DPB Fi12
AATTACCTTTTCCAGGGACG
SEQ ID NO: 290


 


DPB Fi13
AATTACGTGTACCAGTTACG
SEQ ID NO: 291


 


DPB Fi14
AATTACGCGTACCAGTTACG
SEQ ID NO: 292


 


DPB Ri11
CGGCCTCGTCCAGCTCGTAG
SEQ ID NO: 293


 


DPB Ri12
TGGGCCCGCCCAGCTCGTAG
SEQ ID NO: 294


 


DPB Ri13
TGGGCCCGACCAGCTCGTAG
SEQ ID NO: 295


 


DPB Ro
GGACTCGGCGCTGCAGGGTC
SEQ ID NO: 296


 


DPA Fo
AGGAGCTGGGGCCATCAAGG
SEQ ID NO: 297


 


DPA Fi
GACCATGTGTCAACTTATGC
SEQ ID NO: 298


 


DPA Ri1
CTCAGGGGGATCGTTGGTGG
SEQ ID NO: 299


 


DPA Ri2
CTCAGGGGGATCATTGGCGG
SEQ ID NO: 300


 


DPA Ro
CAGCTCCACAGGCTCCTTGG
SEQ ID NO: 301


 


DQB Fo
ACTCTCCCGAGGATTTCGTG
SEQ ID NO: 302


 


DQB Fi
TGTGCTACTTCACCAACGGG
SEQ ID NO: 303


 


DQB Ri1
ACCTCGTAGTTGTGTCTGCA
SEQ ID NO: 304


 


DQB Ri2
AACTGGTAGTTGTGTCTGCA
SEQ ID NO: 305


 


DQB Ro1
ACTCTCCTCTGCAGGATCCC
SEQ ID NO: 306


 


DQB Ro2
ACTCGCCGCTGCAAGGTCGT
SEQ ID NO: 307


 


DQB Ro3
ACTCTCCTCTGCAAGATCCC
SEQ ID NO: 308


 


DQA Fo
TGGTGTAAACTTGTACCAGT
SEQ ID NO: 309


 


DQA Fi
ACCCATGAATTTGATGGAGA
SEQ ID NO: 310


 


DQA Ri
GGAACCTCATTGGTAGCAGC
SEQ ID NO: 311


 


DQA Ro
ACTTGGAAAACACTGTGACC
SEQ ID NO: 312

## Claims

1. A method for producing an immune status profile for at least one human or animal subject comprising the steps of (a) isolating RNA from the subject's white blood cells, (b) amplifying the RNA using multiplex RT-PCR in a first amplification reaction using nested target-specific forward and reverse primers, the forward primers comprising forward inner and forward outer primers and the reverse primers comprising reverse inner and reverse outer primers, the forward and reverse inner primers comprising additional nucleotides to incorporate into at least one resulting amplicon at least one binding site for at least one common primer, (c) rescuing amplicons from the first amplification reaction, (d) amplifying, by the addition of common primers to a second amplification reaction, the amplicons of the first amplification reaction having at least one binding site for a common primer, and (f) sequencing the amplicons of the second amplification reaction using high-throughput sequencing to identify antibody and/or receptor rearrangements in the cells.