Title:
METHOD FOR EVALUATING AND COMPARING IMMUNOREPERTOIRES
Kind Code:
A1


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.



Inventors:
Han, Jian (Huntsville, AL, US)
Application Number:
14/673446
Publication Date:
09/17/2015
Filing Date:
03/30/2015
Assignee:
HAN JIAN
Primary Class:
International Classes:
C12Q1/68
View Patent Images:



Foreign References:
WO2006110855A22006-10-19
Other References:
Arstila et al., Science 286:958-961, October 1999
GenBank GI:36871 (October 1996)
Primary Examiner:
SCHULTZ, JAMES
Attorney, Agent or Firm:
MAYNARD COOPER & GALE PC (Huntsville) (HUNTSVILLE, AL, US)
Claims:
Now, therefore, the following is claimed:

1. A method for producing an immune status profile for a human and/or animal, the method comprising: (a) amplifying, in a first amplification using target specific primers, at least one DNA from a sample of white blood cells from a human or animal subject to produce at least one first amplicon, at least a portion of the target-specific primers comprising additional nucleotides to incorporate into the at least one first amplicon a binding site for a common primer; (b) rescuing the at least one first amplicon from the first amplification; (c) amplifying, in a second amplification using at least one common primer, the at least one first amplicon to produce at least one second amplicon; and (d) sequencing the at least one second amplicon to identify and quantify one or more DNA sequences representing rearrangements to create an immune status profile.

2. The method of claim 1 further comprising the steps of (e) inputting the one or more DNA sequences into a database to provide data which may be stored on a computer, server, or other electronic storage device, (f) inputting a data set of identifying information and characteristics for an individual corresponding to the sequences as data which may be stored on a computer, server, or other electronic storage device, and (g) evaluating the data of step (e) and step (f) for one or more individuals to determine whether a correlation exists between the one or more sequences and one or more characteristics of the individual corresponding to the one or more sequences.

3. The method of claim 1 wherein the target-specific primers are 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.

4. A method for producing an immune response profile, the method comprising: amplifying DNA from a human or animal subject using target-specific primers chosen from among the groups 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.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. patent application Ser. No. 12/425,310, filed Apr. 16, 2009, which claims priority to U.S. provisional patent application No. 61/045,586, filed Apr. 16, 2008, the contents of each are incorporated by reference in their entirety.

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 germline 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 method 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, or Major Histocompatibility Complex (MHC) molecules I or II.

In another aspect of the invention, the method may also comprise an comprising 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

The disclosure can be better understood with reference to the following drawings.

The elements of the drawings are not necessarily to scale relative to each other, emphasis instead being placed upon clearly illustrating the principles of the disclosure.

Furthermore, like reference numerals designate corresponding parts throughout the several views.

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 CLL, and (d) Ig lambda chain in a blood sample from a patient with systemic lupus erythematosis (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 chosed 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, 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, the recombinations in the subpopulation of cells 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 the 454 sequencing method, 454A and 454B primers are linked onto PCR products either 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 at 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 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 cell 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 ordered were ALL-PB-MNC (from 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 (10 ng 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 is some background amplification from the B cell reaction. Further improvement on that primer mix was required to clean 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 μL10×PCR Buffer, 1 μL dNTP, 0.25 μL HotStartTaq Plus, and 39.75 μL H2O. (For a mix of 12 reactions: 60 μL10×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 Fi 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, 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 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. 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 multiplex 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
PrimerSequenceID Number
TRAV1Fo5′-TGCACGTACCAGACATCTGG-3′SEQ ID NO: 1
TRAV1FiAGGTCGTTTTTCTTCATTCCSEQ ID NO: 2
TRAV2FoTCTGTAATCACTCTGTGTCCSEQ ID NO: 3
TRAV2FiAGGGACGATACAACATGACCSEQ ID NO: 4
TRAV3FoCTATTCAGTCTCTGGAAACCSEQ ID NO: 5
TRAV3FiATACATCACAGGGGATAACCSEQ ID NO: 6
TRAV4FoTGTAGCCACAACAACATTGCSEQ ID NO: 7
TRAV4FiAAAGTTACAAACGAAGTGGCSEQ ID NO: 8
TRAV5FoGCACTTACACAGACAGCTCCSEQ ID NO: 9
TRAV5FiTATGGACATGAAACAAGACCSEQ ID NO: 10
TRAV6FoGCAACTATACAAACTATTCCSEQ ID NO: 11
TRAV6FiGTTTTCTTGCTACTCATACGSEQ ID NO: 12
TRAV7FoTGCACGTACTCTGTCAGTCGSEQ ID NO: 13
TRAV7FiGGATATGAGAAGCAGAAAGGSEQ ID NO: 14
TRAV8FoAATCTCTTCTGGTATGTSCASEQ ID NO: 15
TRAV8FiGGYTTTGAGGCTGAATTTASEQ ID NO: 16
TRAV9FoGTCCAATATCCTGGAGAAGGSEQ ID NO: 17
TRAV9FiAACCACTTCTTTCCACTTGGSEQ ID NO: 18
TRAV10FoAATGCAATTATACAGTGAGCSEQ ID NO: 19
TRAV10FiTGAGAACACAAAGTCGAACGSEQ ID NO: 20
TRAV11FoTCTTAATTGTACTTATCAGGSEQ ID NO: 21
TRAV11FiTCAATCAAGCCAGAAGGAGCSEQ ID NO: 22
TRAV12FoTCAGTGTTCCAGAGGGAGCCSEQ ID NO: 23
TRAV12FiATGGAAGGTTTACAGCACAGSEQ ID NO: 24
TRAV13FoACCCTGAGTGTCCAGGAGGGSEQ ID NO: 25
TRAV13FiTTATAGACATTCGTTCAAATSEQ ID NO: 26
TRAV14FoTGGACTGCACATATGACACCSEQ ID NO: 27
TRAV14FiCAGCAAAATGCAACAGAAGGSEQ ID NO: 28
TRAV16FoAGCTGAAGTGCAACTATTCCSEQ ID NO: 29
TRAV16FiTCTAGAGAGAGCATCAAAGGSEQ ID NO: 30
TRAV17FoAATGCCACCATGAACTGCAGSEQ ID NO: 31
TRAV17FiGAAAGAGAGAAACACAGTGGSEQ ID NO: 32
TRAV18FoGCTCTGACATTAAACTGCACSEQ ID NO: 33
TRAV18FiCAGGAGACGGACAGCAGAGGSEQ ID NO: 34
TRAV19FoATGTGACCTTGGACTGTGTGSEQ ID NO: 35
TRAV19FiGAGCAAAATGAAATAAGTGGSEQ ID NO: 36
TRAV20FoACTGCAGTTACACAGTCAGCSEQ ID NO: 37
TRAV20FiAGAAAGAAAGGCTAAAAGCCSEQ ID NO: 38
TRAV21FoACTGCAGTTTCACTGATAGCSEQ ID NO: 39
TRAV21FiCAAGTGGAAGACTTAATGCCSEQ ID NO: 40
TRAV22FoGGGAGCCAATTCCACGCTGCSEQ ID NO: 41
TRAV22FiATGGAAGATTAAGCGCCACGSEQ ID NO: 42
TRAV23FoATTTCAATTATAAACTGTGCSEQ ID NO: 43
TRAV23FiAAGGAAGATTCACAATCTCCSEQ ID NO: 44
TRAV24FoGCACCAATTTCACCTGCAGCSEQ ID NO: 45
TRAV24FiAGGACGAATAAGTGCCACTCSEQ ID NO: 46
TRAV25FoTCACCACGTACTGCAATTCCSEQ ID NO: 47
TRAV25FiAGACTGACATTTCAGTTTGGSEQ ID NO: 48
TRAV26FoTCGACAGATTCMCTCCCAGGSEQ ID NO: 49
TRAV26FiGTCCAGYACCTTGATCCTGCSEQ ID NO: 50
TRAV27FoCCTCAAGTGTTTTTTCCAGCSEQ ID NO: 51
TRAV27FiGTGACAGTAGTTACGGGTGGSEQ ID NO: 52
TRAV29FoCAGCATGTTTGATTATTTCCSEQ ID NO: 53
TRAV29FiATCTATAAGTTCCATTAAGGSEQ ID NO: 54
TRAV30FoCTCCAAGGCTTTATATTCTGSEQ ID NO: 55
TRAV30FiATGATATTACTGAAGGGTGGSEQ ID NO: 56
TRAV34FoACTGCACGTCATCAAAGACGSEQ ID NO: 57
TRAV34FiTTGATGATGCTACAGAAAGGSEQ ID NO: 58
TRAV35FoTGAACTGCACTTCTTCAAGCSEQ ID NO: 59
TRAV35FiCTTGATAGCCTTATATAAGGSEQ ID NO: 60
TRAV36FoTCAATTGCAGTTATGAAGTGSEQ ID NO: 61
TRAV36FiTTTATGCTAACTTCAAGTGGSEQ ID NO: 62
TRAV38FoGCACATATGACACCAGTGAGSEQ ID NO: 63
TRAV38FiTCGCCAAGAAGCTTATAAGCSEQ ID NO: 64
TRAV39FoTCTACTGCAATTATTCAACCSEQ ID NO: 65
TRAV39FiCAGGAGGGACGATTAATGGCSEQ ID NO: 66
TRAV40FoTGAACTGCACATACACATCCSEQ ID NO: 67
TRAV40FiACAGCAAAAACTTCGGAGGCSEQ ID NO: 68
TRAV41FoAACTGCAGTTACTCGGTAGGSEQ ID NO: 69
TRAV41FiAAGCATGGAAGATTAATTGCSEQ ID NO: 70
TRACRoGCAGACAGACTTGTCACTGGSEQ ID NO: 71
TRACRiAGTCTCTCAGCTGGTACACGSEQ ID NO: 72
TRBV1FoAATGAAACGTGAGCATCTGGSEQ ID NO: 73
TRBV1FiCATTGAAAACAAGACTGTGCSEQ ID NO: 74
TRBV2FoGTGTCCCCATCTCTAATCACSEQ ID NO: 75
TRBV2FiTGAAATCTCAGAGAAGTCTGSEQ ID NO: 76
TRBV3FoTATGTATTGGTATAAACAGGSEQ ID NO: 77
TRBV3FiCTCTAAGAAATTTCTGAAGASEQ ID NO: 78
TRBV4FoGTCTTTGAAATGTGAACAACSEQ ID NO: 79
TRBV4FiGGAGCTCATGTTTGTCTACASEQ ID NO: 80
TRBV5FoGATCAAAACGAGAGGACAGCSEQ ID NO: 81
TRBV5aFiCAGGGGCCCCAGTTTATCTTSEQ ID NO: 82
TRBV5bFiGAAACARAGGAAACTTCCCTSEQ ID NO: 83
TRBV6aFoGTGTGCCCAGGATATGAACCSEQ ID NO: 84
TRBV6bFoCAGGATATGAGACATAATGCSEQ ID NO: 85
TRBV6aFiGGTATCGACAAGACCCAGGCSEQ ID NO: 86
TRBV6bFiTAGACAAGATCTAGGACTGGSEQ ID NO: 87
TRBV7FoCTCAGGTGTGATCCAATTTCSEQ ID NO: 88
TRBV7aFiTCTAATTTACTTCCAAGGCASEQ ID NO: 89
TRBV7bFiTCCCAGAGTGATGCTCAACGSEQ ID NO: 90
TRBV7cFiACTTACTTCAATTATGAAGCSEQ ID NO: 91
TRBV7dFiCCAGAATGAAGCTCAACTAGSEQ ID NO: 92
TRBV9FoGAGACCTCTCTGTGTACTGGSEQ ID NO: 93
TRBV9FiCTCATTCAGTATTATAATGGSEQ ID NO: 94
TRBV10FoGGAATCACCCAGAGCCCAAGSEQ ID NO: 95
TRBV10FiGACATGGGCTGAGGCTGATCSEQ ID NO: 96
TRBV11FoCCTAAGGATCGATTTTCTGCSEQ ID NO: 97
TRBV11FiACTCTCAAGATCCAGCCTGCSEQ ID NO: 98
TRBV12FoAGGTGACAGAGATGGGACAASEQ ID NO: 99
TRBV12aFiTGCAGGGACTGGAATTGCTGSEQ ID NO: 100
TRBV12bFiGTACAGACAGACCATGATGCSEQ ID NO: 101
TRBV13FoCTATCCTATCCCTAGACACGSEQ ID NO: 102
TRBV13FiAAGATGCAGAGCGATAAAGGSEQ ID NO: 103
TRBV14FoAGATGTGACCCAATTTCTGGSEQ ID NO: 104
TRBV14FiAGTCTAAACAGGATGAGTCCSEQ ID NO: 105
TRBV15FoTCAGACTTTGAACCATAACGSEQ ID NO: 106
TRBV15FiAAAGATTTTAACAATGAAGCSEQ ID NO: 107
TRBV16FoTATTGTGCCCCAATAAAAGGSEQ ID NO: 108
TRBV16FiAATGTCTTTGATGAAACAGGSEQ ID NO: 109
TRBV17FoATCCATCTTCTGGTCACATGSEQ ID NO: 110
TRBV17FiAACATTGCAGTTGATTCAGGSEQ ID NO: 111
RBV18FoGCAGCCCAATGAAAGGACACSEQ ID NO: 112
TRBV18FiAATATCATAGATGAGTCAGGSEQ ID NO: 113
TRBV19FoTGAACAGAATTTGAACCACGSEQ ID NO: 114
TRBV19FiTTTCAGAAAGGAGATATAGCSEQ ID NO: 115
TRBV20FoTCGAGTGCCGTTCCCTGGACSEQ ID NO: 116
TRBV20FiGATGGCAACTTCCAATGAGGSEQ ID NO: 117
TRBV21FoGCAAAGATGGATTGTGTTCCSEQ ID NO: 118
TRBV21FiCGCTGGAAGAAGAGCTCAAGSEQ ID NO: 119
TRBV23FoCATTTGGTCAAAGGAAAAGGSEQ ID NO: 120
TRBV23FiGAATGAACAAGTTCTTCAAGSEQ ID NO: 121
TRBV24FoATGCTGGAATGTTCTCAGACSEQ ID NO: 122
TRBV24FiGTCAAAGATATAAACAAAGGSEQ ID NO: 123
TRBV25FoCTCTGGAATGTTCTCAAACCSEQ ID NO: 124
TRBV25FiTAATTCCACAGAGAAGGGAGSEQ ID NO: 125
TRBV26FoCCCAGAATATGAATCATGTTSEQ ID NO: 126
TRBV26FiATTCACCTGGCACTGGGAGCSEQ ID NO: 127
TRBV27FoTTGTTCTCAGAATATGAACCSEQ ID NO: 128
TRBV27FiTGAGGTGACTGATAAGGGAGSEQ ID NO: 129
TRBV28FoATGTGTCCAGGATATGGACCSEQ ID NO: 130
TRBV28FiAAAAGGAGATATTCCTGAGGSEQ ID NO: 131
TRBV29FoTCACCATGATGTTCTGGTACSEQ ID NO: 132
TRBV29FiCTGGACAGAGCCTGACACTGSEQ ID NO: 133
TRBV30FoTGTGGAGGGAACATCAAACCSEQ ID NO: 134
TRBV30FiTTCTACTCCGTTGGTATTGGSEQ ID NO: 135
TRBCRoGTGTGGCCTTTTGGGTGTGGSEQ ID NO: 136
TRBCRiTCTGATGGCTCAAACACAGCSEQ ID NO: 137
TRDV1FoTGTATGAAACAAGTTGGTGGSEQ ID NO: 138
TRDV1FiCAGAATGCAAAAAGTGGTCGSEQ ID NO: 139
TRDV2FoATGAAAGGAGAAGCGATCGGSEQ ID NO: 140
TRDV2FiTGGTTTCAAAGACAATTTCCSEQ ID NO: 141
TRDV3FoGACACTGTATATTCAAATCCSEQ ID NO: 142
TRDV3FiGCAGATTTTACTCAAGGACGSEQ ID NO: 143
TRDCRoAGACAAGCGACATTTGTTCCSEQ ID NO: 144
TRDCRiACGGATGGTTTGGTATGAGGSEQ ID NO: 145
TRGV1-5FoGGGTCATCTGCTGAAATCACSEQ ID NO: 146
TRGV1-5,AGGAGGGGAAGGCCCCACAGSEQ ID NO: 147
8Fi
TRGV8FoGGGTCATCAGCTGTAATCACSEQ ID NO: 148
TRGV5pFiAGGAGGGGAAGACCCCACAGSEQ ID NO: 149
TRGV9FoAGCCCGCCTGGAATGTGTGGSEQ ID NO: 150
TRGV9FiGCACTGTCAGAAAGGAATCCSEQ ID NO: 151
TRGV10FoAAGAAAAGTATTGACATACCSEQ ID NO: 152
TRGV10FiATATTGTCTCAACAAAATCCSEQ ID NO: 153
TRGV11FoAGAGTGCCCACATATCTTGGSEQ ID NO: 154
TRGV11FiGCTCAAGATTGCTCAGGTGGSEQ ID NO: 155
TRGCRoGGATCCCAGAATCGTGTTGCSEQ ID NO: 156
TRGCRiGGTATGTTCCAGCCTTCTGGSEQ ID NO: 157

TABLE 2
Sequence
PrimerSequenceID Number
IgHV1aFoAGTGAAGGTCTCCTGCAAGGSEQ ID NO: 158
IgHV1bFoAGTGAAGGTTTCCTGCAAGGSEQ ID NO: 159
IgHV1aFiAGTTCCAGGGCAGAGTCACSEQ ID NO: 160
IgHV1bFiAGTTTCAGGGCAGGGTCACSEQ ID NO: 161
IgHV1cFiAGTTCCAGGAAAGAGTCACSEQ ID NO: 162
IgHV1dFiAATTCCAGGACAGAGTCACSEQ ID NO: 163
IgHV2FoTCTCTGGGTTCTCACTCAGCSEQ ID NO: 164
IgHV2FiAAGGCCCTGGAGTGGCTTGCSEQ ID NO: 165
IgHV3aFoTCCCTGAGACTCTCCTGTGCSEQ ID NO: 166
IgHV3bFoCTCTCCTGTGCAGCCTCTGGSEQ ID NO: 167
IgHV3cFoGGTCCCTGAGACTCTCCTGTSEQ ID NO: 168
IgHV3dFoCTGAGACTCTCCTGTGTAGCSEQ ID NO: 169
IgHV3aFiCTCCAGGGAAGGGGCTGGSEQ ID NO: 170
IgHV3bFiGGCTCCAGGCAAGGGGCTSEQ ID NO: 171
IgHV3cFiACTGGGTCCGCCAGGCTCCSEQ ID NO: 172
IgHV3dFiGAAGGGGCTGGAGTGGGTSEQ ID NO: 173
IgHV3eFiAAAAGGTCTGGAGTGGGTSEQ ID NO: 174
IgHV4FoAGACCCTGTCCCTCACCTGCSEQ ID NO: 175
IgHV4FiAGGGVCTGGAGTGGATTGGGSEQ ID NO: 176
IgHV5FoGCGCCAGATGCCCGGGAAAGSEQ ID NO: 177
IgHV5FiGGCCASGTCACCATCTCAGCSEQ ID NO: 178
IgHV6FoCCGGGGACAGTGTCTCTAGCSEQ ID NO: 179
IgHV6FiGCCTTGAGTGGCTGGGAAGGSEQ ID NO: 180
IgHV7FoGTTTCCTGCAAGGCTTCTGGSEQ ID NO: 181
IgHV7FiGGCTTGAGTGGATGGGATGGSEQ ID NO: 182
IgHJRoACCTGAGGAGACGGTGACCSEQ ID NO: 183
IgHJ1RiCAGTGCTGGAAGTATTCAGCSEQ ID NO: 184
IgHJ2RiAGAGATCGAAGTACCAGTAGSEQ ID NO: 185
IgHJ3RiCCCCAGATATCAAAAGCATCSEQ ID NO: 186
IgHJ4RiGGCCCCAGTAGTCAAAGTAGSEQ ID NO: 187
IgHJ5RiCCCAGGGGTCGAACCAGTTGSEQ ID NO: 188
IgHJ6RiCCCAGACGTCCATGTAGTAGSEQ ID NO: 189
IgKV1FoTAGGAGACAGAGTCACCATCSEQ ID NO: 190
IgKV1FiTTCAGYGRCAGTGGATCTGGSEQ ID NO: 191
IgKV2FoGGAGAGCCGGCCTCCATCTCSEQ ID NO: 192
IgKV2aFiTGGTACCTGCAGAAGCCAGGSEQ ID NO: 193
IgKV2bFiCTTCAGCAGAGGCCAGGCCASEQ ID NO: 194
IgKV3-7FoGCCTGGTACCAGCAGAAACCSEQ ID NO: 195
IgKV3FiGCCAGGTTCAGTGGCAGTGGSEQ ID NO: 196
IgKV6-7FiTCGAGGTTCAGTGGCAGTGGSEQ ID NO: 197
IgKV4-5FiGACCGATTCAGTGGCAGCGGSEQ ID NO: 198
IgKCRoTTCAACTGCTCATCAGATGGSEQ ID NO: 199
IgKCRiATGAAGACAGATGGTGCAGCSEQ ID NO: 200
IgLV1aFoGGGCAGAGGGTCACCATCTCSEQ ID NO: 201
IgLV1bFoGGACAGAAGGTCACCATCTCSEQ ID NO: 202
IgLV1aFiTGGTACCAGCAGCTCCCAGGSEQ ID NO: 203
IgLV1bFiTGGTACCAGCAGCTTCCAGGSEQ ID NO: 204
IgLV2FoCTGCACTGGAACCAGCAGTGSEQ ID NO: 205
IgLV2FiTCTCTGGCTCCAAGTCTGGCSEQ ID NO: 206
IgLV3aFoACCAGCAGAAGCCAGGCCAGSEQ ID NO: 207
IgLV3bFoGAAGCCAGGACAGGCCCCTGSEQ ID NO: 208
IgLV3aFiCTGAGCGATTCTCTGGCTCCSEQ ID NO: 209
IgLV3bFiTTCTCTGGGTCCACCTCAGGSEQ ID NO: 210
IgLV3cFiTTCTCTGGCTCCAGCTCAGGSEQ ID NO: 211
IgLV4FoTCGGTCAAGCTCACCTGCACSEQ ID NO: 212
IgLV4FiGGGCTGACCGCTACCTCACCSEQ ID NO: 213
IgLV5FoCAGCCTGTGCTGACTCAGCCSEQ ID NO: 214
IgLV5FiCCAGCCGCTTCTCTGGATCCSEQ ID NO: 215
IgLV6FoCCATCTCCTGCACCCGCAGCSEQ ID NO: 216
IgLV7-8FoTCCCCWGGAGGGACAGTCACSEQ ID NO: 217
IgLV9,CTCMCCTGCACCCTGAGCAGSEQ ID NO: 218
11Fo
IgLV10FoAGACCGCCACACTCACCTGCSEQ ID NO: 219
IgLV6, 8FiCTGATCGSTTCTCTGGCTCCSEQ ID NO: 220
IgLV7FiCTGCCCGGTTCTCAGGCTCCSEQ ID NO: 221
IgLV9FiATCCAGGAAGAGGATGAGAGSEQ ID NO: 222
IgLV10-CTCCAGCCTGAGGACGAGGCSEQ ID NO: 223
11Fi
IgLC1-7RoGCTCCCGGGTAGAAGTCACTSEQ ID NO: 224
IgLC1-7RiAGTGTGGCCTTGTTGGCTTGSEQ ID NO: 225

TABLE 3
PrimerSequenceSequence ID Number
HLAI Fo11CCCACTCCATGAGGTATTTCSEQ ID NO: 226
HLAI Fo12CCTACTCCATGAGGTATTTCSEQ ID NO: 227
HLAI Fo31GCGGGGAGCCCCGCTTCATCSEQ ID NO: 228
HLAI Fo32GCGGGAAGCCCCGCTTCATCSEQ ID NO: 229
HLAI Fo33GTGGAGAGCCCCGCTTCATCSEQ ID NO: 230
HLAI Fo34GCGGAAAGCCCCGCTTCATCSEQ ID NO: 231
HLAI Fo35GCGGAAAGCCCCACTTCATCSEQ ID NO: 232
HLAI Fo36GCGGGAAGCCCCACTTCATCSEQ ID NO: 233
HLAI Fi11GTGGGCTACGTGGACGACACSEQ ID NO: 234
HLAI Fi12GTGGGCTACGTGGACGGCACSEQ ID NO: 235
HLAI-Fi21GTTCGTGCGGTTCGACAGCGSEQ ID NO: 236
HLAI-Fi22GTTCGTGCGGTTTGACAGCGSEQ ID NO: 237
HLAI-Fi23GTTCGTGAGGTTCGACAGCGSEQ ID NO: 238
HLAI Ri11TAATCCTTGCCGTCGTAGGCSEQ ID NO: 239
HLAI Ri12TAATCCTTGCCGTCGTAAGCSEQ ID NO: 240
HLAI Ri13TAATCTTTGCCGTCGTAGGCSEQ ID NO: 241
HLAI Ro11GGTCCTCGTTCAGGGCGATGSEQ ID NO: 242
HLAI Ro12GGTCCTCTTTCAGGGCGATGSEQ ID NO: 243
HLAI Ro13GGTCCTCGTTCAAGGCGATGSEQ ID NO: 244
HLAI Ro14GATCCTCGTTCAGGGCGATGSEQ ID NO: 245
HLAI Ro15GGTCCTCATTCAGGGCGATGSEQ ID NO: 246
HLAI-Fo41GCCTACGACGGCAAGGATTASEQ ID NO: 247
HLAI-Fo42GCTTACGACGGCAAGGATTASEQ ID NO: 248
HLAI-Fo43GCCTACGACGGCAAAGATTASEQ ID NO: 249
HLAI-Fi31CATCGCCCTGAACGAGGACCSEQ ID NO: 250
HLAI-Fi32CATCGCCCTGAAAGAGGACCSEQ ID NO: 251
HLAI-Fi33CATCGCCTTGAACGAGGACCSEQ ID NO: 252
HLAI-Fi34CATCGCCCTGAACGAGGATCSEQ ID NO: 253
HLAI-Fi35CATCGCCCTGAATGAGGACCSEQ ID NO: 254
HLAI-Ri21GGTATCTGCGGAGCCCGTCCSEQ ID NO: 255
HLAI-Ri22GGTATCTGCGGAGCCACTCCSEQ ID NO: 256
HLAI-Ri23GGTGTCTGCGGAGCCACTCCSEQ ID NO: 257
HLAI-Ri24GGTATCCGCGGAGCCACTCCSEQ ID NO: 258
HLAI-Ro21GCAGCGTCTCCTTCCCGTTCSEQ ID NO: 259
HLAI-Ro22CCAGCTTGTCCTTCCCGTTCSEQ ID NO: 260
HLAI-Ro23CCAGCGTGTCCTTCCCGTTCSEQ ID NO: 261
HLAI-Ro24GCAGCGTCTCCTTCCCATTCSEQ ID NO: 262
HLAI-Ro25GCAGCGTCTCCTTCCKGTTCSEQ ID NO: 263
DRB17 Fo11TGTCATTTCTTCAATGGGACSEQ ID NO: 264
DRB1 Fo12AGTGTCATTTCTTCAACGGGSEQ ID NO: 265
DRB1 Fo13GTGTTATTTCTTCAATGGGASEQ ID NO: 266
DRB1 Fo14GTGTCAATTCTTCAATGGGASEQ ID NO: 267
DRB4 FoGTGTCATTTCCTCAATGGGASEQ ID NO: 268
DRB1 Fi11GGAGCGGGTGCGGTTGCTGGSEQ ID NO: 269
DRB1 Fi12GGAGCGGGTGCGGTACCTGGSEQ ID NO: 270
DRB1 Fi13GGAGCGGGTGCGGTTCCTGGSEQ ID NO: 271
DRB1 Fi14GGAGCGGGTGCGATTCCTGGSEQ ID NO: 272
DRB1 Fi15GGAGCGGGTGCGGTATCTGCSEQ ID NO: 273
DRB1 Fi16GGAGCGGGTGCGGTTACTGGSEQ ID NO: 274
DRB45 FiACATCTATAACCAAGAGGAGSEQ ID NO: 275
DRB6 FiACATCCATAAACGGGAGGAGSEQ ID NO: 276
DRB7 FiTATAACCAAGAGGAGTACGTSEQ ID NO: 277
DRB Ri11AACCCCGTAGTTGTGTCTGCSEQ ID NO: 278
DRB4 RiAACCCCGTAGTTGTGTCTGCSEQ ID NO: 279
DRB6 RiCGTAATTGTATCTGCAGTAGSEQ ID NO: 280
DRB7 RiTAGTTGTCCACTTCGGCCCGSEQ ID NO: 281
DRB Ro1CGCTGCACTGTGAAGCTCTCSEQ ID NO: 282
DRB Ro2CGCTGCACCGTGAAGCTCTCSEQ ID NO: 283
DRA FoCCTGTGGAACTGAGAGAGCCSEQ ID NO: 284
DRA FiCAACGTCCTCATCTGTTTCASEQ ID NO: 285
DRA RiCTGCTGCATTGCTTTTGCGCSEQ ID NO: 286
DRA RoTTACAGAGGCCCCCTGCGTTSEQ ID NO: 287
DPB FoGTCCAGGGCAGGGCCACTCCSEQ ID NO: 288
DPB Fi1AATTACGTGTACCAGGGACGSEQ ID NO: 289
DPB Fi12AATTACCTTTTCCAGGGACGSEQ ID NO: 290
DPB Fi13AATTACGTGTACCAGTTACGSEQ ID NO: 291
DPB Fi14AATTACGCGTACCAGTTACGSEQ ID NO: 292
DPB Ri11CGGCCTCGTCCAGCTCGTAGSEQ ID NO: 293
DPB Ri12TGGGCCCGCCCAGCTCGTAGSEQ ID NO: 294
DPB Ri13TGGGCCCGACCAGCTCGTAGSEQ ID NO: 295
DPB RoGGACTCGGCGCTGCAGGGTCSEQ ID NO: 296
DPA FoAGGAGCTGGGGCCATCAAGGSEQ ID NO: 297
DPA FiGACCATGTGTCAACTTATGCSEQ ID NO: 298
DPA Ri1CTCAGGGGGATCGTTGGTGGSEQ ID NO: 299
DPA Ri2CTCAGGGGGATCATTGGCGGSEQ ID NO: 300
DPA RoCAGCTCCACAGGCTCCTTGGSEQ ID NO: 301
DQB FoACTCTCCCGAGGATTTCGTGSEQ ID NO: 302
DQB FiTGTGCTACTTCACCAACGGGSEQ ID NO: 303
DQB Ri1ACCTCGTAGTTGTGTCTGCASEQ ID NO: 304
DQB Ri2AACTGGTAGTTGTGTCTGCASEQ ID NO: 305
DQB Ro1ACTCTCCTCTGCAGGATCCCSEQ ID NO: 306
DQB Ro2ACTCGCCGCTGCAAGGTCGTSEQ ID NO: 307
DQB Ro3ACTCTCCTCTGCAAGATCCCSEQ ID NO: 308
DQA FoTGGTGTAAACTTGTACCAGTSEQ ID NO: 309
DQA FiACCCATGAATTTGATGGAGASEQ ID NO: 310
DQA RiGGAACCTCATTGGTAGCAGCSEQ ID NO: 311
DQA RoACTTGGAAAACACTGTGACCSEQ ID NO: 312