WO2009051672A2 - Natural killer immunoglobulin-like receptor (kir) assay - Google Patents

Abstract

The present invention provides methods, assays and enabling compositions for genotyping natural killer (NK) cells by immunoglobulin-like receptor (KIR) expression. In particular, the present invention allows for KIR expression profiling in, for example, samples from donors and patients, results of which are useful in transplantation therapies involving NK cells.

Description

NATURAL KILLER IMMUNOGLOBULIN-LIKE RECEPTOR (KIR) ASSAY

FIELD OF THE INVENTION

The present invention provides methods and assays and enabling compositions for genotyping natural killer (NK) cells according to the type of immunoglobulin-like receptors (KIR) they express. In particular, the present invention allows for KIR expression profiling in, for example, samples from recipients and donors of allogeneic bone marrow and hematopoietic stem cell transplants, wherein NK cells are expected to be therapeutic for recipients if the matching of donor NK cells to recipient is optimized for that purpose.

BACKGROUND OF THE INVENTION

Several families of highly diversified molecules govern tolerance to "self and immunity against "non-self in humans and animals. Examples are the blood groups {e.g., ABO blood group system), the human leukocyte antigen (HLA) system, T- lymphocyte antigen receptors (T-cell receptors), and B-lymphocyte antigen receptors.

In general, transplanting ("grafting") a population of cells from a donor who is immunologically non-identical to the recipient (or "host") exposes the host's immune system to an array of foreign antigens, with the result that fragments (generally, small peptides) from at least some of the donor cells find their way to so-called major histocompatibility complex molecules, or "MHCs" (referred to, for historical reasons, as human leukocyte antigens or "HLAs"), arrayed on the surfaces of host cells. Each fragment, when complexed with an MHC molecule, becomes an antigen that can interact with a receptor on a host T-cell. The interaction sets into motion a process whereby the body's immune system acquires the ability to recognize any future encounter with the antigen as a signal that more of the donor cells have invaded. When such an encounter occurs, an elaborate defense is mounted against the invading cells until the host has destroyed ("rejected") them.

As endogenous T-cells mature, they interact with a multitude of (autologous) MHC molecules (whether or not complexed to a foreign fragment). Immature T-cells bearing receptors that react with an autologous MHC molecule, will not mature. An individual's mature T-cells therefore tend to fit the individual's repertoire of MHC molecules in a "self-self manner. When an individual's T-cell population is obliterated and then re-constituted from a colony of immunologically non-identical donor stem cells, the T-cell receptor repertoire of the T-cell population that descends from the colony, assuming that the mismatch doesn't lead to outright rejection, also comes to conform to the host's HLA genotype, leaving behind only a residuum of chimeras.

Other molecules that participate in immunity include receptors found on natural killer (NK) cells. NK cells are polymorphonuclear leukocytes (their nuclei take many forms). Interferons, other cytokines, and the Fc portion of antibodies activate NK cells, which thereupon release enzymes that induce apoptosis in nearby cells, especially virus- infected cells, cancer cells and allogeneic ("foreign") cells. Like T-cells, NK cells are lymphocytes sensitive to HLAs, but their tolerance of "self ' antigens arises from a quite different mechanism. Two broad classes of receptors on NK cells modulate the activation of NK cells. One class is lectin-like. The other, found in humans, is immunoglobulin-like (the "KIRs"), a reference to the structural similarity of these receptors to immunoglobulin molecules. The polymorphism of the genes that encode these receptors rivals that of the HLAs that bind to them (Carrington et al. Curr. Top. Microbiol Immunol. 298: 225-257, 2006). About one-half of all KIRs, upon binding an HLA, inhibit (that is, make refractory to cytokines and antibodies) the NK cells on which they are displayed. They are "iKIRs." Binding of an HLA to the others ("aKIRs") tends to activate (or at least fails to inhibit) the cell. Whether a particular NK cell is active or inhibited appears to depend upon the balance between iKIR interactions and aKIR interactions.

Unlike a population of T-cells re-constituted from a colony of stem cells, wherein a less than perfectly matched T-cell receptor repertoire becomes less donor-like over time, a re-constituted population of NK cells carries a KIR repertoire that remains highly reflective of the donor's repertoire (Vilches et al. Annu. Rev. Immunol. 20: 217-251, 2002). "Mismatched" donor KIRs therefore interact with host HLAs at a high frequency. Nevertheless, a self-non-self reaction is unlikely: Each NK cell displays its own "mini- repertoire" of iKIRs. In general, if even one of those iKIRs interacts with an HLA on a host cell, the killer cell will remain quiescent (Borrego et al. MoI. Immunol. 38: 637-660, 2001), leaving the host cell undisturbed. An NK cell mounts a destructive response when it encounters a cell that is broadly deficient in expressed HLAs. Thus, NK cells often tolerate non-self, but not "missing self."

Therapeutic obliteration of bone marrow and of hematopoietic stem cells, followed by transplantation of "new" marrow or stem cells, are established methods for treating hematologic diseases such as leukemias. Since T cell receptors and the HLA molecules with which they interact are both highly polymorphic, clinical hematopoietic cell transplantation predictably involves "mismatches" in T cell receptor type and in HLA type between allogeneic donors and recipients. If grossly mismatched (unrelated donor- recipient pair), graft rejection or graft-vs-host disease (GVHD) can be expected. Less severe mismatches (siblings), on the other hand, can be therapeutic for the leukemic patient. One of the reasons that cancer cells can grow, multiply, and spread is that the body accepts them as "self." Thus, cancer cells tend not to alert the body's immune defenses. Transplanted, unmatched T-cells, on the other hand, do not accept the host's cancer cells as self. A destructive interaction, called the graft versus tumor effect, results. The effect appears to be most powerful in diseases that progress slowly, like chronic leukemia, low-grade lymphoma, and in some cases multiple myeloma, but is weaker in the rapidly growing acute leukemias. The therapeutic aspect of transplanting incompletely matched blood products is most apparent when leukemia returns ("relapse") in a treated patient and a follow-up dose of the original donor's white blood cells is administered: The donated cells sharply combat the leukemic cells immunologically, which keeps the leukemic cell population from expanding.

NK cells transplanted into a non-identical leukemic host can also be curative. Because any given NK cell displays a plurality of iKIR types, and healthy host cells display a plurality of HLAs, at least one NK cell-inhibiting iKIR:HLA interaction is likely. Leukemic host cells, however, are HLA-deficient. The probability that a host's leukemic cells will inhibit the host's NK cells is therefore reduced. Thus, transplanted NK cells would appear to be well-suited to counter leukemias, particularly if their aKERs do not match the aKIRs of their host (Ruggeri et al. Transpl. Immunol. 14: 203-206, 2005). The data, however, present a mixed picture. KIR-KIR repertoire mismatch appears to offer no significant benefit for survival of leukemia patients, or for prevention of relapse after chemotherapy, or in the incidence of GvHD (Koenecke et al. Exp. Hematol. 31: 911-923, 2003; Gagne et al. Human Immunol. 63: 911-923, 2003). Bignon et al. (Curr. Opinion Immunol. 16: 634-643, 2004) reported contrary results, but in related host-donor pairs only. Recipients of transplants from unrelated donors experienced an increased incidence of graft failure. In pediatric leukemia patients "rescued" with human stem cell transplants after ablative therapy, KIR mismatch decreases relapse rate (Leung et al. J. Immunol. 172: 644-650, 2004; Hsu et al. Blood 105: 4878-4884, 2005) but no such benefit is seen in patients with SCID (Keller et al. J. Clin. Immunol. 27: 109-116, 2007). It is likely that full KIR genotyping could reveal the basis for these divergent results and lead, ultimately, to a thorough understanding of how to conduct therapeutic mismatching in an optimal manner. Accordingly, what are needed are methods and systems that allow practitioners and investigators to more accurately and efficiently determine the KIR genotype of a sample. Such methods and systems would provide tools for use in transplant therapeutics and in KIR-related research.

SUMMARY OF THE INVENTION

The present invention provides compositions, methods and assays for genotyping natural killer (NK) cells according to their expression of immunoglobulin-like receptors (KIRs). In particular, embodiments of the present invention allow for KTR expression profiling in, for example, samples from donors and patients, results of which are useful in optimizing transplantation therapies involving NK cells.

In some embodiments, the invention comprises a set of oligonucleotide-primer pairs, the set configured to amplify amplicons of KIRs such that each amplicon is specific to a KIR family, wherein the length of each specific amplicon is different. In some embodiments, the present invention provides methods and assays for the specific detection of KIRs in donor and recipient samples {e.g., blood samples) using mRNA expression typing. The present invention provides amplification primers in such combination as to result in short amplicons {e.g., shorter than 500bp), each of a length specific for a particular KIR family and, at the same time, amenable to visualization {e.g., using fluorescently labeled primers) by means of instruments {e.g., genetic sequence analyzers), especially instruments wherein the amplifications can be performed and the amplicons displayed in a multiplex fashion. In some embodiments, the incorporation of a means of measuring CD56 mRNA or other marker that is specific to NK cells provides for normalization of the samples for NK cell numbers. According to certain embodiments, the assembled set of primers are provided in a kit, which kit preferably includes a means for identifying a specific NK-cell marker, and reagents, enzymes and buffers that enable the assays to be conducted.

In some embodiments, the present invention provides methods and assays for performing individual RT-PCR or other amplification reactions, preferably using primers selected according to criteria provided by the present invention, on mRNA from NK cells for determining KIR expression. In some embodiments, the present invention provides methods and assays for conducting the amplification of a plurality of mRNA species {e.g. multiple amplification reactions in one tube) using primers of the present invention.

In some embodiments, the present invention provides for the visualization of the products of the amplification reactions, hi some embodiments, an aliquot of one RT- PCR or other amplification reaction is loaded into one well (or other single point of origin) in a separation medium {e.g., an agarose or polyacrylamide gel), thereby allowing for the visualization of one amplification reaction per well, wherein the separation medium may be configured as a slab or column and may be disposed in an electric, magnetic or gravitational field, hi other embodiments, the products of separate amplification reactions are combined into a mixture or complex {i.e., "multiplexed") and loaded into one well or point of origin, thereby allowing for the visualization of multiple amplification reaction products emanating from a single point of origin into the separation medium. In some embodiments, the amplification reactions themselves are conducted simultaneously on a plurality of mRNA species as a complex in a single reaction vessel and an aliquot of the products of the multiplex amplification reaction is loaded into one well for separation and visualization as a spectrum of amplification products. hi one aspect, the invention is embodied as a method of assembling or constructing a set of oligonucleotide-primer pairs wherein at least one forward KIR oligonucleotide primer is provided, and a plurality of reverse KIR oligonucleotide primers is provided, wherein each reverse KIR primer is specific for one and only one KIR family, and each forward KIR primer and reverse KIR primer defines an amplicon having a nucleotide length less than about 500 nucleotides and each such nucleotide length is different. In assembling the set, one may add an amplification control by providing an oligonucleotide-primer pair consisting of a forward cell-marker primer and a reverse cell-marker primer wherein the primer pair defines a transcript of an NK cell marker such as CD56. It is advantageous to provide forward KTR primers that are labeled, preferably with a fluorescent molecule.

In one aspect, the invention is embodied in a method for determining the expression of a KTR family in a sample. According to the method, a nucleic acid sample from a population of polymorphonuclear cells is provided. At least one pair of oligonucleotide primers is applied to the sample, wherein one member of the pair is selected from the group consisting of SEQ ID NO. 15 and SEQ ID NO. 16 and one member is selected from the group consisting of SEQ ID NO. 1 through SEQ ID NO. 14. The nucleic acid sample is amplified with the oligonucleotide primers and, based on the amplification, the presence or absence of expression of the KIR family in the sample is determined.

The invention is also embodied in a method for determining the expression of a plurality of KIR families in a sample. According to this method a nucleic acid sample from a population of polymorphonuclear cells is provided, to which sample is applied a set of oligonucleotide primers. The set is assembled with at least one forward KIR oligonucleotide primer, together with a plurality of reverse KIR oligonucleotide primers, wherein each reverse KIR primer is specific for one and only one KIR family. Each forward KER. primer and reverse KTR primer defines an amplicon having a nucleotide length less than about 500 nucleotides and each such nucleotide length is different. Amplicons of the nucleic acid sample are generated and used to determine the KIR families expressed in the sample. In a preferred embodiment, the amplicons are generated in a single reaction vessel. In a most preferred embodiment, the amplicons are separated from one another in a single separation step.

In one embodiment, the invention provides an assay for tissue typing blood products for KIR genotype. The assay can be part of a procedure for determining tissue compatibility for use in transplantation therapy. DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1 shows an exemplary electropherogram of KJR spectratypes in a mixed PBMNC sample from healthy donors. The sample was tested by using a single PCR for each KIR gene. Each peak represents a unique KIR gene fragment. The size value is shown on the x-axis and the peak height on the y-axis.

Figure 2 shows an exemplary experiment demonstrating KIR expression in donors after stem cell mobilization and selection. Each KIR gene family is shown on the x-axis and its expression level on the y-axis. KJR expression was quantified before mobilization (grey) after mobilization (open) and after stem cell selection (hatched).

Figure 3 shows an exemplary electropherogram of KIR spectratypes in two mixed PCR populations. The size value is shown on the x-axis and the peak high on the y-axis. Each peak represents one KIR gene fragment.

DEFINITIONS

Certain embodiments of the invention described herein are enabled by means of the polymerase chain reaction ("PCR"). In general, PCR is an enzyme-catalyzed process for generating many copies of a region of a nucleotide polymer. The region is said to be "amplified." Amplification relies in part on selecting a short pair of sequences in the polynucleotide of interest, which sequences bind to or "hybridize" specific short

"oligonucleotides. With the appropriate reagents and enzymes, PCR makes copies (or the complements thereof) of the region of the longer polymer that is defined between the binding sites, resulting in an exponentially expanded population of so-called "amplicons." The oligonucleotides "prime" the reaction. Typically, a primer pair consists of a primer having a sequence that complements a sequence running "forward" in the larger polymer and a primer that complements a sequence running in the "reverse" direction. The allowable length of the region between the primers can be varied widely, but lengths of less than 500 consecutive nucleotides (the "nucleotide length") are preferred in embodiments of the invention. The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g.,

RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, expression of a protein of interest in a host cell, expression of a ribozyme, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).

As used herein, the term "protein of interest" refers to a protein encoded by a nucleic acid of interest. As used herein, the term "exogenous gene" refers to a gene that is not naturally present in a host organism or cell, or is artificially introduced into a host organism or cell.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e.g., proinsulin). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences. The sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through "translation" of mRNA. Gene expression can be regulated at many stages in the process. "Up- regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g. , transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence thus codes for the amino acid sequence.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-A-G-T-S','' is complementary to the sequence "3'-T- C-A-5'." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. The terms "homology" and "percent identity" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology (i.e., partial identity) or complete homology (i.e., complete identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe (i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest) will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize {i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

As used herein, the term "T_m" is used in reference to the "melting temperature" of a nucleic acid. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid

Hybridization [1985]). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T_m.

As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. "High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42⁰C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PCvH₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed. "Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed. "Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄^H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [5OX Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.

The terms "in operable combination," "in operable order," and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. It is advantageous for some embodiments of the invention and the various applications thereof to identify the expression products of the genes that encode KIRs rather than the genes themselves as they are encoded in the DNA (desoxyribonucleic acid) of chromosome 19. Expressions of the gene-code are conveniently detected when they are first transcribed from the DNA as so-called messenger RNA (ribonucleic acid). These "mRNA" transcripts presage the actual receptors that will appear as proteins embedded in the surfaces of NK cells. In general, each NK cell displays several KIR family members, but each cell does not necessarily display the same set. The full spectrum, across all NK cells, comprises an individual's KIR "repertoire." As used herein, "repertoire" may be used interchangeably with "genotype," although practitioners will understand that the former term generally refers to phenotype (i.e., a trait or set of traits) while the latter refers to the combination of alleles located on homologous chromosomes that determines phenotype. In the case of KIRs, at least 14 such alleles are known. For each, there are many variations of the code (the genes are "polymorphic") so each of the 14 is really a "family." As used herein, the term "KIR" may refer to a single member of one of the families or to the entire family, as the context so admits. As used herein, the term "expression profiling" refers generally to a method of assessing the genotype (in this case the KIR genotype) of an individual by detecting the presence or absence of the family- specific mRNAs in a sample under inspection. Expression profiling yields data useful in "expression typing." As used herein, the term relates to procedures for tissue typing, that is, determining the compatibility or incompatibility of a donor tissue with a host in connection with tissue transplantation.

The polymerase chain reaction preferred for use herein is the so-called reverse transcriptase PCR or RT-PCR, wherein mRNA transcripts are enzymatically converted to DNA and then the DNA is subjected to PCR. The method is not limited to these means for determining the KIR repertoire of an individual or a sample suspected of containing KIRs. In general, a "sample" as used herein relates to a sample of blood obtained from a donor or prospective donor of an organ or tissue, including but not limited to blood or elements thereof (e.g., lymphocytes), collectively referred to as "blood products." The recipient of a donor's organ or tissue, typically a patient (or subject), may also be referred to herein as a "host."

The term "multiplex" as used herein relates to two or more events that can take place at separate times or in separate spaces being made to take place at the same time and space. Thus, a plurality of amplification reactions could be conducted, each in its own reaction vessel. If they are conducted at once in a single vessel, the reactions are said to be multiplexed. Similarly, the analysis, visualization or measurement of the products of two or more amplification reactions could be analyzed, visualized or measured separately in time or space (e.g, electrophoresed in separate lanes of a gel). If such analyses, visualizations or measurements are conducted at once on a single gel lane, the analysis is said to be multiplexed. In the context of certain embodiments of the invention, multiplexing is a means of displaying or visualizing amplification products as a spectrum. In particular, when a sample of blood from a patient, for example, is subjected to a multiplex amplification reaction using a set of primers assembled according to the invention and the mixed products of the reaction are separated from one another by electrophoresis, the amplification product of each KIR family in the patient's KIR repertoire travels to its own site in the separation medium, so that the repertoire is visualized as a spectrum. Each patient has a patient-specific spectrum, which is referred to herein as a "spectratype."

DETAILED DESCRIPTION OF THE INVENTION

Killer cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by natural killer cells. The KIR genes are homologous but highly polymorphic. They are found in a cluster on chromosome 19ql3.4 within the 1 Mb leukocyte receptor complex (LRC). The gene content of the KIR gene cluster varies among haplotypes, although several 'framework' genes are found in all haplotypes (KIR3DL3, KTR3DL1, KIR3DL4, KIR3DL2). KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain. KIR proteins with the long cytoplasmic domain transduce inhibitory (iKIR) signals upon ligand binding via an immune tyrosine-based inhibitory motif (ITIM), while KIR proteins with the short cytoplasmic domain lack the ITIM motif and instead associate with the tyrosine kinase binding protein TYRO to transduce activating signals (aKIR). The ligands for several KIR proteins are subsets of HLA molecules; thus, KIR proteins are thought to play an important role in regulation of the immune response.

When it comes to transplantation therapies involving NK cells, such as transplantation therapies for leukemias {e.g., bone marrow transfusions and hematopoietic stem cell transplantation), the matching (or, perhaps more to the point, the strategic "mismatching") of NK cells from donor to recipient may be advantageous in ameliorating relapse and graft vs host disease (GvH). The explicit use of NK cells for therapeutic purposes, however, will preferably employ methods that provide accurate, convenient and reliable determinations of the KIR repertoire of both donor and recipient.

NK cells are called "natural" because of the role they play in the innate immune response (Carrington and Martin, 2006, Curr Topics Micro Immuno 298:225-257). KIRs are also expressed on a minor subset of memory/effector T-cells that participate in acquired immunity. The KIR repertoire of NK cells is shaped in subtle ways by the HLA environment to which the cells are exposed (Shilling et al., Blood 101:3730-3740, 2003), but is by no means determined by an individual's HLA repertoire as T-cell receptors are (Vilches and Parham, Ann Rev Immunol 20:217-251, 2002). The KIR gene repertoire is diverse, being determined not only by gene locus but also by allelic polymorphism. With such extensive diversity, it is uncommon for two individuals to have the same KIR gene repertoire. iKIRs regulate natural killer (NK) cell function by recognizing self-HLA ligands that belong to Class I of the major histocompatibility complex. Recognition leads to NK cell inhibition, which prevents NK cells from killing healthy autologous cells.

Meanwhile, aKTRs recognize non-self HLAs to initiate NK-cell activity against infected or transformed cells (Carrington et al. Curr. Top. Microbiol. Immunol. 298: 225-257, 2006; Borrego et al, MoI Immunol 38:637-660, 2001). The "missing self hypothesis assumes that NK activation depends upon a balance between inhibitory and activating signals such that a cell that cannot present sufficient self-signals to an NK cell will tip the balance toward NK activation, especially if that NK cell is picking up non-self signals at the same time. The diversity of the KIR repertoire and the balance between KIRs and their ligands affects susceptibility to many diseases such as autoimmune, inflammatory diseases and malignancy. In recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT),

NK cells do not cause GvH disease even when donor and recipient are mismatched, because the graft NK cells attack only mismatched host cells that express little or no HLA class-I, leukemic cells being a prominent example (Ruggeri et al., Transplant Immunol 14:203-206, 2005). These advantages have made NK-cell treatment attractive as an adjunct in allo-HSCT. Available information about KIR genotypes derives largely from investigations directed at identifying KIR genotypes that correlate with disease or untoward post- transplant effects. It is known that the genotype is not fully expressed transcriptionally or phenotypically even in healthy individuals (Leung et al., J Immunol 174:6540-6545, 2005). There is evidence that donor KIR genes may be down-regulated during hematologic reconstitution after HSCT, particularly when myeloablative conditioning is used (Shilling et al, Blood 101:3730-3740, 2003). And, when the appearance of KIR transcripts is delayed post-transplant, the risk of acute GvH increases (Denis et al., Hum Immunol 66:447-459, 2005). Taken together, these results suggest that KIR expression undergoes kinetic changes that may affect the development of disease and the outcome of treatment. However, KIR expression in the healthy population, in donors during stem cell recruitment and selection, and in patients during the peri-transplant period all remain to be investigated.

Commercial products are available for KIR typing, such as the QIAGEN® Olerup SSP Kits for KIR, DYNAL® KIR Genotyping SSP Kit, and the Miltenyi Biotec, Inc. KIR Typing Kit. All these products utilize amplification methods for typing KIRs present in a sample followed by agarose gel electrophoresis of the single amplifications. Other systems for KIR typing are reported (Lin et al., Am. J. Pathol. 159:1671-1679, 2001; Uhrberg, Immunity 7:753-763, 1997; Leung et al, J. Immunol. 174:6540-6545, 2005; Leung et al., Immunol 172:644-650, 2004; Shilling et al, Blood 101:3730-3740, 2003; and U.S. Patent Applications 2004/0259103 and 2005/019695). However, none of the commercial products or published references disclose primers that, when combined in multiple pairs, would allow KIR amplifications to be multiplexed so that amplifications of diverse KIRs are performed in one tube and the amplicons separated from one another when run from one well of a gel. Such advances would allow for greater ease of use and increased efficiency of time, resources, and reagents than currently available options.

Embodiments of the present invention provide such methods. One embodiment comprises a set of KIR polymerase chain reaction (PCR) primers designed to quantitatively determine repertoire expression. In one aspect, the invention comprises the criteria by which the set is designed. These criteria can be used to admit or deny entry of primer pairs to the repertoire-determining set for purposes of expanding the set to accommodate any newly discovered KIRs. The criteria include, (1) the specificity of each primer pair must be unambiguous; (2) each primer pair of the set must define an amplicon of a nucleotide length amenable to amplification in an automated system; (3) the length of the amplicon each primer pair defines must be sufficiently different for each primer pair that each amplicon can be resolved from the others in a suitable separation step such as gel electrophoresis, and (4) a means of amplifying an expression product unique to NK cells must be provided.

For example, a set of KIR primers that includes only two fluorescently-labeled common forward primers and 14 reverse primers specific to each KIR family was generated. With this design, each specific primer not only hybridizes to a specific KIR transcript, but also generates fluorescently-labeled PCR fragments, each having its own distinct length, that are easily recognized and quantified by a DNA genetic analyzer. The PCR fragments were designed to be approximately 148-329 bp to conform to a preferred size range less than 500 bp. The two common forward primers simplify the PCR procedure and provide the opportunity to further reduce the number of PCR reactions by generating mixed PCR products (e.g., multiplex PCR) in which the distinct length of PCR fragments is retained (Figure 3). This design also reduces the expense of fluorescent- labeled primers. Further, a transcript of CD56, the expression of which is determinative of NK cells (i.e., it is a "cell marker"), is amplified as an endogenous control (or "amplification control") to effectively reduce error by providing an index of the number of NK cells in the sample.

In some embodiments, KIR primers were designed according to the sequence profile described by Garcia et al., Immunogenetics 55:227-239, 2003. To confirm the specificity of the primers, PCR products generated by each of the reverse (RV) primers in combination with a forward (FW) primer were sequenced. The specific sequence was found in the PCR products of all primer pairs except 3DL3, for which no positive signal in any of the tested healthy donors and patients was found. Each pair of KIR primers generated a specific PCR fragment with distinct length as demonstrated on an electropherogram (Figure 1). In some embodiments, the present invention provides methods and systems for

KIR genotyping. In some embodiments, the methods and systems comprise fluorescently labeled forward primers in operable combination with unlabeled reverse primers, wherein each pair of primers amplifies one KTR family. In some embodiments, the methods and systems of the present invention further comprise reagents, buffers, enzymes, and other solutions, proteins and the like for performing RT-PCR and/or PCR reactions. In some embodiments, the methods and systems comprise an internal amplification control for each amplification reaction. In some embodiments, the amplification control is a CD56 encoding nucleic acid. In some embodiments, methods and systems further comprise reagents, buffers, enzymes, and the like for performing polyacrylamide gel electrophoresis. In some embodiments, the methods and systems of the present invention are used in conjunction with a genetic sequence analyzer, such as an ABI PRISM®

Genetic Analyzer. However, the present invention is not limited by the instrumentation used in performing the amplification and/or visualization reaction and it is contemplated that any instrument capable of performing one or both of amplification and/or visualization of an amplified product is amenable for use with the methods of the present invention.

In preferred embodiments, the label affixed to a forward primer is fluorescent. Such fluorescent labels include, but are not limited to, 6-FAM, fluorescein isothiocyanate, phycoerythrin, allophycocyanin, Cy 3 and Cy 5. Dyes useful in labeling primers (e.g., directly or indirectly) are known to those skilled in the art, and can be found in, for example, the Handbook of Fluorescent Probes and Research Chemicals,

Richard P. Haugland, Molecular Probes, Inc. In some embodiments, the combinations of forward and reverse primers provide for amplification products less than 500bp, less than 400bp, less than 350bp. In some embodiments, the combinations of primers provides for amplification products preferably from about lOObp to about 400bp, from about 120bp to about 380bp, from about 140bp to about 350bp, or from about 148bp to about 329bp.

In some embodiments, a KER amplification reaction is performed as a single amplification reaction, wherein only one set of KIR primers is present in one tube or one well of a 96 well plate or other plate format used for amplification purposes. In some embodiments, several sets of KIR primers are present in one tube or one well of a plate, such that multiple KER amplification are performed at one time (e.g., a multiplex reaction). In some embodiments, an aliquot of a KIR amplification reaction is visualized on a polyacrylamide gel, such that one KIR reaction is run in one well of a polyacrylamide gel. In some embodiments, aliquots of separate KIR amplification reactions are combined and visualized on a polyacrylamide gel, such that multiple reaction products are run in one well of a polyacrylamide gel for visualization. In some embodiments, an aliquot of a KIR multiplex reaction is visualized on a polyacrylamide gel, such that all products of a multiplex reaction are run in one well of a polyacrylamide gel for visualization. Multiplexing of amplification reactions and of the separation process required to visualize a complete spectrum is enabled with the primer set of Table 4. In some embodiments, the present invention provides methods and systems for typing the KIR genotype of a donor and/or a recipient blood sample contemplated for use in transplantation therapy. In some embodiments, the present invention is used in combination with other diagnostic assays for tissue typing blood products, such as methods and assays found in US Patents 5,256,543, 5,420,013, 5,702,885, 6,670,124, and 5,972,604 (all incorporated herein by reference in their entireties). As such, embodiments of the present invention include determining the compatibility of a donor sample for use by a recipient for transplantation therapy, for example, for leukemic diseases and other blood related disorders where NK cells and or stem cells are used for transplantation. KIR expression has not previously been described in leukemia patients. In developing embodiments of the present invention, reduced expression of the majority of KIR families in leukemia patients before transplantation, especially in patients with ALL and NHL, was found. Some patients tested showed an imbalance of iKIR and aKTR. It was found that patients with a higher iKIR/aKIR ratio before transplantation had a substantially higher rate of relapse. Therefore, the pre-transplant iKIR/aKIR ratio of the recipient is important to determine as experiments performed in developing embodiments of the present invention demonstrate that a higher ratio is indicative of relapse and the outcome of the transplantation. As such, in some embodiments the present invention provides a diagnostic in determining relapse of a patient following HSCT. EXPERIMENTATION

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. After confirming the specificity of the primers, assays were used to assess KIR expression in healthy donors and leukemia patients before HSCT. The impact of stem cell mobilization and haplo-HSCT were also examined in donors and their recipients.

It was found that with successful engraftment, most patients demonstrated a 100% donor KIR pattern one-month post-HSCT, regardless of the conditioning regimen. This pattern is stable at 12 months post-transplant. In some cases, recipients' KIR patterns were not consistent with their peripheral blood chimerism post-HSCT. However, in one case, a mixed donor/recipient KIR pattern persisted for one year post-HSCT despite 100% donor peripheral blood chimerism at one month and donor dominance. As such, NK cell chimerism is not completely parallel to peripheral blood chimerism. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that the mechanism responsible for iKIR/aKIR balance in the recipient is dominant after transplantation, even when the donor KIR pattern is expressed. If iKIR dominates aKIR excessively, aKIR initiation of NK-cell killing is impeded, thereby increasing the risk of an adverse post-transplant outcome. In developing embodiments of the present invention, KIR gene expression was determined in healthy donors (in two groups; 1-20 years and 21-51 years of age). Ranges of KIR gene expression in the healthy donors tested are shown in Table 1.

Table 1

Age group Age group

KIR gene 1-2Oy 21-51y p value

(n=50) (n=37)

2DS4 19.9±3.3 16.3±3.5 0.12

mean percentage.

The group of 21-51 years of age showed significantly lower mean expression of six KJR families (2DLl, 2DlA, 2DSl, 2DS3, 3DSl, 3DL2) than the younger group. Four of those KIR families were aKIR. The mean ratio of lKJR (2DLl, 2DL2, 2DL3, 2DL5, 3DLl and 3DL2) to aKIR (2DL4, 2DSl, 2DS2, 2DS3, 2DS4, 2DS5 and 3DS1) was significantly (p=0.037) higher in 21-51 years group than that in younger group. The overall KIR expression decreased as age increased. A statistically significant inverse correlation between age and expression was seen in 2DLl, 2DL4, 2DS3 and 3DSl. There was no statistically significant difference in mean KIR expression or mean iKJR/aKIR ratio between male and female donors. Thirty-two categories of KTR expression profile in the healthy donors was observed. The 2DLl, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4, 3DLl, and 3DL2 KIR families were frequently expressed (Table 2). Table 2

Age 2DLl 2DL2 2DL3 2DL4 2DL5 2DSl 2DS2 2DS3 2DS4 2DS5 3DLl 3DSl 3DL2

1-20

100% 94% 96% 100% 50% 48% 90% 36% 100% 46% 92% 52% 100%

p va ue NA 0 63 0 NA 0 19 O 67 1 0 0 16 NA 0.38 0.4 0_03 NA

Four families (2DL5, 2DS3, 2DS5 and 3DS 1) were less frequent in the older group, but only 3DSl was significantly lower. Again, three of the families were aKIR. KIR spectratyping profiles of healthy donors were compared with their KIR phenotypes as determined by flow cytometry. There was no qualitative difference between these phenotypes and the KLR gene expression profiles.

In developing embodiments of the present invention, KJR repertoire in pediatric leukemic patients (acute lymphoblastic leukemia (ALL), 18; acute myelogenous leukemia (AML), 9; chronic myelogenous leukemia (CML), 8; non-Hodgkin lymphoma (NHL), 2) before allo-HSCT was examined. Lower expression of 11 of the 14 KIR families (11/14) was found in leukemic pediatric patients as compared to healthy donors of the same age range. The patients with ALL or NHL were most affected, although the disease subgroups were too small to be compared statistically. The mean ratio of iKIR/aKIR was higher in the patient group but the increase was not statistically significant (p=0.25).

Experimentation was performed to determine whether the pre-HSCT iKJR/aKIR ratio was related to relapse and post-transplant survival. Patients with an iKIR/aKIR ratio >2.0 (2.0 is the mean ratio +SD for healthy donors) showed a higher rate of relapse (40%) than did patients with a ratio <2.0 (18.5%), however the difference was not statistically significant (p=0.49). The 1-year survival rate did not differ between the two ratio groups.

Patient samples were tested to determine whether there was a change in donor KLR expression after mobilization and selection of stem cells. In healthy donors, the majority of KIR families (2DL2, 2DL3, 2DL4, 2DS3, 2DS4, 2DS4, 3DLl, 3DSl and 3DL2) showed increased expression (p>0.05) after stem cell mobilization, but the iKIR/aKIR ratio did not change significantly. After stem cell selection, the expression of most KIR families returned to their pre-mobilization levels (Figure 2).

Further experimentation was performed to determine if there existed a change in KIR expression in recipients after HSCT. It was determined that KIR expression increased in recipients after transplantation. Five of 12 patients showed a donor KIR repertoire pattern one-month post haplo-HSCT when their peripheral blood chimerism was 100% donor (Table 3).

Table 3

D donor KIR pattern, R. recipient KIR patten

and recipient

Three patients had a mixed donor-recipient KIR pattern with their donor after 1 month when their peripheral blood chimerism was 100% donor, but two patients had their donor pattern at month 3 post-transplant. Four patients had the same KIR pattern as their donors before and after HSCT. One patient had a mixed donor-recipient KIR pattern for an entire year post-HSCT whereas the patient's peripheral blood chimerism ranged from 93% to 100% donor.

The distribution of KIR genotypes in the healthy population has been broadly studied and 37 KIR haplotypes have been described (Carπngton and Martin, 2006) However, the KTR expression pattern in the healthy population has not been reported In developing embodiments of the present invention, 32 categoπes of KIR expression patterns in healthy donors were found, suggesting a wide vaπation in KIR expression in healthy individuals. Similarly, is it was found that of the KIR genotypes, 2DLl, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4, 3DLl, and 3DL2 were expressed frequently. However, three aKIRs (2DS3, 2DS5 and 3DS 1) were expressed less frequently in adult donors. An overall inverse correlation was found between KIR gene expression and age that affected mainly the activating KIR families. Therefore, it is demonstrated herein that aKTR expression declines quantitatively as well as qualitatively with age. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that that this decline may delay activation of the NK killing function. The overall quantity of iKIR is higher than that of aKIR in the healthy population, suggesting the dominance of the self-protective iKIR function. Subjects and transplant regimens

To establish a reference range, KIR gene expression was examined in 50 healthy sibling donors whose ages ranged from 1 to 20 years matching the patient age range. Thirty-seven healthy adult donors 21 to 51 years of age were also studied. A total of 126 samples were obtained from 37 patients pre- transplantation and 12 donor-recipient pairs during stem cell processing and post-transplant. All 12 patients received CD3 depleted haplo-HSCT. Six of these patients were conditioned with a reduced-intensity regimen comprising fludarabine, thiotepa, melphalan and OKT3 without total-body irradiation (TBI) and antithymocyte globulin (ATG). Mycophenolate mofetil was used for GvHD prophylaxis. The other six patients received myeloablative conditioning with TBI (12Gy), cyclophosphamide, thiotepa, ATG and OKT3. Cyclosporine was given for GvHD prophylaxis. KIR assay KIR assays consisted of RT-PCR and fragment electrophoresis on a genetic sequence analyzer. Total RNA (lμg) was purified from IO⁶ Ficoll-enriched peripheral blood mononuclear cells (PBMNCs) by using the RNeasy Mini Kit (QIAgen Inc, Valencia, CA). Complementary DNA (cDNA) was synthesized by using Superscript II reverse transcriptase and random hexamer primers (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. PCR was performed in a volume of 25μl containing 1/200 of the cDNA (corresponding to approximately 5ng original RNA), IX AmpliTaq Gold Master Mix (Applied Biosystems, Branchburg, New Jersey), and 25OnM of 1 of 14 specific reverse (RV) primers combined with 1 of 2 forward (FW) primers conjugated to the fluorescent dye 6-FAM (Table 4). The 2DFW-FAM primer (SEQ ID NO. 15) was used with the RV primers 2DLl RV (SEQ ID NO. 1), 2DL2RV (SEQ ID NO. 2), 2DL3RV (SEQ ID NO. 3), 2DS IRV (SEQ ID NO. 6), 2DS2RV (SEQ ID NO. 7), 2DS3RV (SEQ ID NO. 8), 2DS4RV (SEQ ID NO. 9) and 2DS5RV (SEQ ID NO.10). The 2DL4-3DFW-FAM primer (SEQ ID NO. 16) was used with RV primers 2DL4RV (SEQ ID NO. 4), 2DL5RV (SEQ ID NO. 5), 3DL1RV (SEQ ID NO. 11), 3DL2RV (SEQ ID NO. 12), 3DL3RV (SEQ ID NO. 13) and 3DS1RV (SEQ ID NO. 14).

Table 4

Length

KIR gene family Sequence of PCR fragment (bp)

ill

CAATGAGGTGCAAAGTGTCCTTAT 196

2DS4RV CTCTCCAATGAGGTGCAAAGTGTT

lliilii 2DFW-FAM ATGGCGTGTGTTGGGTTCTTC

The CD56-mRNA-encoding marker was used as an endogenous control to normalize for input RNA and to calculate the percentile of each KIR family in the total CD56 transcripts (using forward amplification primerCD56W-FAM (SEQ ID NO. 17) and reverse amplification primer CD56RV (SEQ ID NO. 18)). The PCR conditions were 95°C for 6 min followed by 35 cycles of 95 ⁰C for 15 sec, 58 °C for 60 sec. and a final extension step of 72 ⁰C for 5 min. PCR products (4μl) were diluted in 1 Oμl of 1 X TE buffer and mixed with 20μl of formamide/size standard mixture (20μl 500LIZ size standard per ml of formamide). The final mixture was denatured at 95⁰C for 7 min and chilled to 4⁰C for more than 2 min before analysis on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Data were collected and analyzed by Genemapper Software version 3.7. All samples were measured in duplicate PCR reactions.

The percent expression of each KIR gene was calculated as follows:

The quantity of peak area for a KIR transcript

KIR gene expression % =

The quantity of peak area for CD56 transcript Flow cytometric analysis of KIR phenotypes

Peripheral blood was drawn in VACUTAINER™ tubes containing trisodium citrate, citric acid and dextrose (ACD). The KIR phenotype was determined by using a 6- COLOR BD™ LSRII Flow Cytometer (Becton Dickinson, San Jose, CA). Whole blood was incubated with the monoclonal antibodies and red cells were lysed with an ammonium chloride-based reagent. The monoclonal antibodies used were anti-CD45- FITC (fluorescein isothiocyanate), anti-CD3-ECD (Phycoerythrin-Texas Red), anti-CD5- APC (allophycocyanin), anti-CD 14- APC-Cy7-A, anti-CD 158a,h-(EB6)-PE

(Phycoerythrin), anti-CD 158b-FITC (CH-L), and anti-NKBl-(DX9)-FITC. Collected data are analyzed using FACS DIVA™ software or FLOWJO™ software. The live cell population was discriminated by using DAPI (Invitrogen, Carlsbad, CA).

The CD14-negative population (equivalent to the lymphocyte population) was selected and gated for the CD56 positive/CD3 negative population. This cell population was analyzed on the basis of KIR 2DLl (Clone EB6) positive vs. KIR 2DL2/3 (Clone CH-L) negative; KIR3DL1 (Clone DX9) negative; KIR 3DLl (Clone DX9) positive vs. KJR 2DL2/3 (Clone GL 183) negative; KIR 2DLl (Clone EB6) negative; KJR 2DL2/3 (CH-L) positive vs. KIR 2DLl (Clone EB6) negative; IQR 3DLl (Clone DX9) negative. Results were calculated as absolute cell numbers. Data were analyzed by using FACS DIVA software or FLOWJO software. Quantitative analysis of chimerism

As previously described (Chen et al., 2005, Blood 105:886-893) donor and recipient alleles were discriminated on the basis of short tandem repeat (STR) PCR with markers established for genetic fingerprinting on the ABI Prism 3100 Genetic Analyzer (Applied Biosystems Incorporated, Foster City, CA). Sequencing

To examine the specificity of the KIR primers, PCR products generated by each pair of FW and RV primers were sequenced. PCR product (50μl) was purified by using a QIAquick PCR purification kit (QIAGEN, Maryland USA) and was sequenced by using BIG DYE® Terminator (v3.1 ) Chemistry on an ABI Prism 3730XL DNA analyzer. Statistical analyses

KIR expression levels were compared by using the Mann Whitney U test. Fisher's exact test was used to compare the frequency of KIR expression. Univariate regression models were used to assess the relationship between KER expression levels and age. In the model, each category of KTR expression level was used as a dependent variable and age was used as the independent variable.

Survival was measured from the date of allo-HSCT to the date of death or date of last follow-up. The probability of overall survival was estimated by using Kaplan Meier method and the associated standard error was calculated by the method of Peto and Pike. The cumulative incidence rate of relapse was calculated by the method of Kalbfleisch and Prentice. Survival curves for patients with an iKIR/aKIR ratio <2.0 vs >2.0 were compared by the Mantel-Haenszel test. The statistical significance with a probability of less than 0.05 was considered statistically significant. All analyses were done using SAS software. All publications and patents mentioned in the present application are herein incorporated by reference for all purposes. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A primer set of oligonucleotide-primer pairs configured to amplify fourteen KIR families, said primer pairs comprising:

(a) at least one forward KIR oligonucleotide primer,

(b) fourteen reverse KIR oligonucleotide primers wherein

(i) each said reverse KIR primer is specific for one and only one KIR family, and

(ii) each said forward KIR primer and reverse KIR primer defines an amplicon having a nucleotide length between about 100 nucleotides and 400 nucleotides, wherein the length of each said amplicon is different.

2. The primer set of claim 1 further comprising an amplification control.

3. The primer set of claim 2 wherein said amplification control comprises an oligonucleotide-primer pair consisting of a forward cell-marker primer and a reverse cell-marker primer wherein said primer pair defines a transcript of an NK cell marker.

4. The primer set of claim 3 wherein said NK cell marker is CD56.

5. The primer set of claim 1 wherein said forward KIR primer is labeled.

6. A method for determining the expression of a KIR family in a sample comprising: a) providing a nucleic acid sample from a population of polymorphonuclear cells, b) applying at least one pair of oligonucleotide primers to said sample, wherein one member of said pair is selected from the group consisting of SEQ ID NO. 15 and SEQ ID NO. 16 and one member is selected from the group consisting of SEQ E) NO. 15 and SEQ ID NO. 16 and one member is selected from the group consisting of SEQ ED NO. 1 through SEQ ID NO. 14. c) amplifying said nucleic acid sample with said oligonucleotide primers, d) determining the presence or absence of expression of said KIR family in said sample based on said amplification.

7. A method for determining the expression of a plurality of KIR families in a sample comprising: a) providing

(i) the set of primers of Claim 1, and

(ii) a nucleic acid sample from a population of leukocytes; b) generating amplicons by amplifying said sample with said set; and c) determining the KIR families expressed in said sample based on said amplicons.

8. The method of claim 7 wherein said amplifying is performed in a single reaction vessel.

9. The method of claim 8 wherein said amplicons are separated from one another in a single separation step.

10. An assay for tissue typing blood products for KIR genotype comprising the method of claim 6.

11. A method for determining tissue compatibility for use in transplantation therapy comprising an assay of claim 6.

12. A kit, comprising the primer set of Claim 1.