Tracing dynamic expansion of human <fc>NK</fc>cell subsets by highresolution analysis of <fc>KIR</fc> repertoires and cellular differentiation

Natural killer (NK) cells are key cellular components of the innate immune system that act at the interface between innate and adaptive immune responses [1]. An increasing body of evidence shows that specific clones of NK cells may be expanded in vivo under the influence of viruses such as human cytomegalovirus (CMV) [2, 3]. These adaptive-like NK-cell responses have been proposed to represent a human counterpart to the NK-cell memory responses observed in mice [4], and seem to be driven by activating receptors, including NKG2C and activating killer cell immunoglobulin-like receptors (KIRs) [2, 5, 6]. So far, clonal-like expansion of specific NK-cell subsets has been documented mostly in the context of primary CMV infection, or conditions that are linked to a clinical or subclinical reactivation of CMV [2, 3, 6–9]. Even so, there is an increasing interest in mapping adaptive-like NK-cell responses in other acute or chronic infections as well as in cancer. The dynamic expansion and functional tuning (education) of NK cells are modulated by activating and inhibitory KIRs interacting with polymorphic determinants (KIR ligands) on HLA class I molecules [10, 11]. Expression of distinct KIRs at the cell surface on T and NK cells is stochastic and is influenced by variations in gene copy number and sequence [12–15]. Therefore, analysis of KIR repertoires on populations of T and NK cells by flow cytometry across a wide range of HLA and KIR backgrounds represents a significant challenge. Protocols for such analysis must overcome intrinsic limitations in available reagents, cross-reactivity of monoclonal antibodies (mAbs) due to the high degree of similarity between KIR gene products and unexpected staining patterns resulting from KIR gene polymorphisms [16, 17]. Here, we describe recently developed staining procedures and an optimized workflow to accurately analyze the human KIRome using flow cytometry and the implementation of this protocol in the evaluation of adaptive-like NK-cell responses. Our recent analysis of KIR expression on NK cells in 204 healthy individuals in large part employed the strategy outlined below. That study first unveiled a significant proportion of rare staining patterns that precluded a standard down-stream analysis by Boolean gating in the software [2]. Genetic testing revealed that most of these patterns were caused by the previously described unusual binding patterns of specific anti-KIR antibodies to allelic variants of KIR2DL3, such as KIR2DL3*005 and KIR2DL3*015 [17]. To accommodate these atypical expression patterns in the analysis of NK-cell repertoires, a refined 15-color flow cytometry panel and a flowchart with sequential quality controls (QCs) was developed (Fig. 1 and Supporting Information Fig. 1). This system enabled us to verify the presence or absence of specific KIRs at the cell surface. As shown in the flow chart (Supporting Information Fig. 1), the outlined strategy can be implemented in the absence of high-resolution KIR genotyping; however, typing all individuals for their KIR gene content is highly recommended. The first QC is based on combining the anti-KIR antibodies EB6 (anti-KIR2DL1/S1) and GL183 (antiKIR2DL2/L3/S2). In Figure 1A, five typical KIR expression patterns are shown. Whereas donors #1 and #2 display normal expression patterns and can be subjected to a standard Boolean gating strategy, donors #3, #4, and #5 exhibit a diagonal staining pattern as well as multiple populations in the double-positive gate, thus requiring a further QC check before downstream analysis can be undertaken (Fig. 1B and C). Indeed, this staining pattern is the hallmark of the expressed KIR2DL3*005 allele and results in a false-positive signal in the KIR2DL2/S2 and KIR2DS1 gates, respectively (Fig. 1D and E) [17]. As a reference material for correct interpretation of staining patterns, we provide the staining and relevant genotypes for 54 healthy donors (Supporting Information Fig. 2). In the absence of KIR genotyping, the decision to include donors with peculiar staining patterns in downstream KIR repertoire analysis can be based on the results of QC2 and QC3. These QCs allow for identification and exclusion of donors with KIR2DL3*005+ NK cells co-expressing KIR2DL2/S2 and/or KIR2DS1, since the latter KIRs cannot be distinguished from KIR2DL3*005 with currently available mAbs. For donors passing QC1, the Boolean gating is straightforward as exemplified for one typical haplotype A/A and one typical haplotype B/X donor (Fig. 1D). However,