KIR/HLA Genotyping

KIR Genotyping

Killer-cell immunoglobulin-like receptors (KIR) govern the killing decisions of natural killer (NK) cells and, by recognizing HLA class I, shape their alloreactivity — making them core variables for donor selection and immunogenetic characterization in allogeneic / off-the-shelf CAR-NK and cell therapy. We provide KIR/HLA genotyping spanning gene content, copy number, allele-level genotypes and haplotypes, with two complementary strategies for different needs — hybrid-capture next-generation sequencing and long-read (third-generation) sequencing — delivering molecular-level evidence for donor selection, Master Cell Bank (MCB) / starting-material characterization, and IND/BLA regulatory submissions.

1. What Is KIR

Natural killer (NK) cells are part of innate immunity and decide whether to kill in real time through a set of germline-encoded activating and inhibitory receptors; the killer-cell immunoglobulin-like receptor (KIR) family is the core receptor set that recognizes HLA class I. KIRs are either long-tailed (inhibitory, recruiting SHP-1/2 via cytoplasmic ITIMs to suppress killing) or short-tailed (activating, signaling through DAP12); the net result of these signals sets the NK activation threshold. The KIR locus lies at chromosome 19q13.4, spans 100–350 kb, carries 4–20 genes per haplotype with more than 600 known alleles, and combines centromeric (Cen) and telomeric (Tel) halves into group A and group B haplotypes; a 0–4 “B-content score” is calculated from the number of Cen/Tel B-motifs. This structural and sequence complexity makes KIR one of the most difficult regions of the human genome to genotype precisely.

2. KIR × HLA: Why Joint Typing Is Essential

KIR can be thought of as the “key” and HLA class I as the “lock”: an inhibitory KIR is “read” and transmits an inhibitory signal only when its corresponding HLA ligand is present on the cell surface. Common pairings include KIR2DL1–HLA-C2, KIR2DL2/2DL3–HLA-C1, KIR3DL1–HLA-Bw4 and KIR3DL2–HLA-A3/A11. However, KIR (chromosome 19) and HLA (chromosome 6) are independent and unlinked — typing KIR without knowing the HLA ligand cannot establish which “brakes” actually apply. KIR must therefore be typed and interpreted together with HLA class I, which is why both of our strategies account for HLA class I. It should be noted that associations between KIR/HLA and transplant or treatment outcomes are correlative, disease-specific evidence and do not constitute a prediction or guarantee of product efficacy.

3. Clinical and Translational Value

The immunogenetic diversity of KIR has shown value in clinical and translational research. In transplantation: a KIR-ligand mismatch in the donor-versus-recipient direction releases the corresponding NK subset from inhibition and produces alloreactivity, associated with reduced leukemia relapse (Ruggeri et al., 2002); selecting unrelated donors by the B-content score improves outcomes in acute myeloid leukemia (AML), with a disease-specific effect (Cooley et al., 2010). In cell therapy: cord-blood-derived anti-CD19 CAR-NK has shown encouraging response rates and a favorable safety profile in early-phase trials (Liu et al., 2020); the natural missing-self recognition of NK cells and their lower GvHD risk make them well suited to off-the-shelf products, for which the donor KIR/HLA background is an important part of immunogenetic characterization.

4. Resolution Tiers

KIR genotyping can be delivered at three progressively higher resolution tiers: gene content (presence/absence of each KIR gene, for donor screening and the B-content score) → copy number / CNV (adding duplication/deletion information on top of gene content) → allele-level plus centromeric/telomeric haplotype (resolving specific alleles and reconstructing complete haplotypes, for confirmation and functional interpretation). Higher resolution carries more information but also places greater demands on the method and sample quality. Choosing a strategy is, in essence, matching the resolution tier to the intended use.

5. Two Detection Strategies

For different resolution needs and throughput scenarios, we offer two complementary strategies that can be used on their own or combined — NGS for screening, long-read for confirmation:

Strategy 1 · Hybrid-Capture NGS: Solution-phase targeted probes enrich the 13 KIR genes and HLA class I in a single reaction; Illumina short-read sequencing with the PING pipeline returns gene content, copy number and allele-level genotypes, with concurrent HLA class I typing. Its strengths are joint KIR/HLA typing and scalability, suited to donor screening and starting-material/population characterization. See the dedicated page: “KIR Genotyping by Hybrid-Capture NGS”.

Strategy 2 · Long-Read Sequencing: Single-molecule long-read sequencing (PacBio HiFi / Oxford Nanopore) reads complete KIR gene sequences directly, resolving highly homologous paralogs, reconstructing complete centromeric/telomeric haplotypes and presenting structural variation. Its strengths are the highest allele-level resolution and complete phase, suited to MCB/starting-material confirmation and confirmation of NGS results. See the dedicated page: “KIR Genotyping by Long-Read Sequencing”.

Figure 1. The two KIR genotyping strategies and the resolution tiers.

6. Choosing a Strategy

7. Applications

Allogeneic / off-the-shelf CAR-NK: immunogenetic characterization of donors and starting materials.

Transplant donor selection: adding KIR to HLA typing to assess the B-content score and the direction of KIR-ligand mismatch.

Starting material / Master Cell Bank (MCB): genetic-background documentation of donor-derived cells in support of release.

Population and cohort studies: large-scale association studies of KIR/HLA genotypes with disease or treatment outcome.

IND/BLA characterization: supporting evidence for donor selection and immunogenetic characterization of cell-therapy products.

8. Regulatory Status

KIR/HLA genotyping is not a mandatory release attribute; it is a scientific input for donor selection and for immunogenetic characterization of the starting material / Master Cell Bank. This service can serve as supporting evidence within the frameworks of the FDA guidance “Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products” (2024), ICH Q5D (derivation and characterization of cell substrates), ICH Q2(R2) (validation of analytical procedures), and China's NMPA “Technical Guidelines for Research and Evaluation of Cell Therapy Products (Trial)” (2017) and CDE “Technical Guidelines for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial)” (2022). The method can be qualified fit-for-purpose or fully validated per ICH Q2(R2) according to the development stage. Detailed parameters, sample requirements and deliverables for each strategy are provided on the corresponding dedicated pages.

9. References

[1] Norman PJ, et al. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing. Am J Hum Genet. 2016;99(2):375–391. doi:10.1016/j.ajhg.2016.06.023

[2] Marin WM, et al. High-throughput Interpretation of KIR Short-read Sequencing Data with PING. PLoS Comput Biol. 2021;17(8):e1008904. doi:10.1371/journal.pcbi.1008904

[3] Downing J, et al. Allele-level KIR genotyping by long-read Oxford Nanopore sequencing. HLA. 2025;106(3):e70400. doi:10.1111/tan.70400

[4] Cooley S, Weisdorf DJ, Guethlein LA, et al. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood. 2010;116(14):2411–2419. doi:10.1182/blood-2010-05-283051

[5] Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002;295(5562):2097–2100. doi:10.1126/science.1068440

[6] Liu E, Marin D, Banerjee P, et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020;382(6):545–553. doi:10.1056/NEJMoa1910607

[7] Milius RP, Mack SJ, Hollenbach JA, et al. Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string. Tissue Antigens. 2013;82(2):106–112.

[8] ICH. Q2(R2): Validation of Analytical Procedures. Step 4 version, 2023.