COMMENTARY

A natural killer cell takes sharp aim at the

world of bacteria COMMENTARY

Peter Parhama,b,1

Killer cell Ig-like receptors (KIR) are expressed on Gibbon-2D natural killer (NK) cells, lymphocytes of innate immu- nity and reproduction. The principal KIR ligands are the polymorphic HLA-A, HLA-B, and HLA-C glycopro- teins of the human major histocompatibility complex. Chimpanzee C1-specific K44 The KIR form 4 phylogenetic lineages. Lineage III KIR are most numerous and include all KIR that recognize epitopes of HLA-C, the most important KIR ligands. Well established is the role of inhibitory lineage III KIR Chimpanzee in educating NK cells to be tolerant of healthy cells C2-specific M44 and ready to kill infected or malignant cells. By comparison, functions for the activating lineage III 2DL7 44M Chimpanzee KIR have proved elusive, particularly for KIR2DS4, the 2DS4 44K and human most common activating KIR. As reported in PNAS (1), 2DS4 44K 2DL9 44E Sim et al. find KIR2DS4 has a binding site with much narrower specificity for HLA-C and peptide than the

inhibitory KIR. In prokaryote genomes, they identify a 2DS2 44K Human highly conserved nonamer sequence in bacterial 2DL3 44K C1-specific 2DL2 44K recombinase A. The corresponding peptide binds 2DS5 44T HLA-C*05:01, forming a ligand that activates human 2DS3 44T C2-specific 2DS1 44M NK cells bearing KIR2DS4. The potential of this li- 2DL1 44M gand−receptor combination is to make NK cell re- sponses against many different bacterial infections. Fig. 1. The diagram summarizes phylogenetic That the KIR2DS4 ligand is now clearly seen to be a relationships between human and chimpanzee lineage III KIR. They form 4 clades. Oldest are the chimpanzee C1- combination of HLA-C*05:01 and a common bacterial specific KIR that have lysine 44. Second oldest are the peptide is a discovery that should encourage and in- chimpanzee C2-specific KIR that have methionine 44. vigorate future research on other orphan KIR. Third oldest is the clade containing human and chimpanzee KIR2DS4, as well as 2 C2-specific receptors: NK Cells Are Controlled by Receptors for MHC one with methionine 44 and the other with glutamate 44. Most recently evolved are all of the 7 other human lineage Class I III KIR, which all derive from a C1-specific progenitor. NK cells were first studied in the context of killing tu- mor cells that escaped CD8 T cell attack by reducing their surface expression of MHC class I (2). During malignant and infected cells. Human NK cells have 2 development, human NK cells acquire activating and unrelated types of inhibitory receptor that recognize inhibitory receptors that recognize variable HLA-A, HLA class I and educate NK cells in complementary HLA-B, and HLA-C, and conserved HLA-E. Inhibitory ways. One is the conserved CD94:NKG2A receptor HLA class I receptors educate developing NK cells (3), that recognizes conserved HLA-E; the other is the di- creating a dynamic intracellular balance between ac- verse family of KIR that recognize highly variable HLA- tivating and inhibitory signals. This balance ensures A, HLA-B, and HLA-C ligands. that NK cells do not disturb healthy cells but promptly KIR recognition of HLA-C focuses on 2 epitopes respond to the lower amounts of HLA class I on defined by dimorphism at positions 77 and 80 of the

aDepartment of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; and bDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305 Author contributions: P.P. wrote the paper. The author declares no conflict of interest. Published under the PNAS license. See companion article 10.1073/pnas.1903781116. 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1907937116 PNAS Latest Articles | 1of3 Downloaded by guest on October 1, 2021 α1 domain. Serine 77 and asparagine 80 define the C1 epitope HLA-C*05:01 has the C2 epitope, the ligand for KIR2DL1. In recognized by inhibitory KIR2DL2 and KIR2DL3, whereas aspar- maturing NK cells, the KIR are expressed stochastically. For agine 77 and lysine 80 define the C2 epitope recognized by individuals who have HLA-C*05:01, KIR2DL1, and KIR2DS4, one KIR2DL1, KIR2DS1, and some KIR2DS5 allotypes (4, 5). In the KIR subset of NK cells expresses KIR2DL1 and KIR2DS4, a second nomenclature, 2D and 3D refer to the number of Ig-like domains. subset expresses KIR2DL1 but lacks KIR2DS4, a third subset ex- Letters L and S refer to the length of the cytoplasmic tail, which presses KIR2DS4 but lacks KIR2DL1, and a fourth subset ex- is Long in the inhibitory receptors and Short in the activating presses neither KIR2DL1 nor KIR2DS4. Sim et al. (1) examined the receptors. interplay between the KIR2DS4 and KIR2DL1 receptors. When NK The C1 and C2 specificities were first distinguished during the 1990s, using cellular assays of NK cell killing (4, 5). Subsequently, As reported in PNAS, Sim et al. find KIR2DS4 has an in vitro binding assay was used, in which KIR-Fc fusion a binding site with much narrower specificity for were tested for binding to panels of beads, each coated with one HLA-A, HLA-B, or HLA-C allotype (6). Although each bead is HLA-C and peptide than the inhibitory KIR. coated with one species of HLA, its bound peptides are very heterogeneous, because the HLA class I was isolated from human cells expressing both KIR2DS4 and KIR2DL1 were cultured with cells and is therefore loaded with thousands of different peptides. target cells expressing complexes of HLA-C*05:01 and P2-AW, This approach defined the precise HLA class I specificities for P2-AY, or P2-AF, the inhibitory signals coming from KIR2DL1 KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS1 and KIR2DS5, but overwhelmed the activating signals transduced by KIR2DS4. In gave no significant reactions with KIR2DS2 and KIR2DS3, and only contrast, the subset of NK cells that expresses KIR2DS4, but not weak heterogeneous reactions with KIR2DS4. Following these KIR2DL1, is activated by complexes of these peptides with HLA- studies in the mid-1990s, KIR2DS2, KIR2DS3, and KIR2DS4 still C*05:01. Unexpectedly, Sim et al. find that NK cells expressing languished as orphan receptors some 20 y later. KIR2DS4, but not KIR2DL1, give a similarly strong response to HLA-C*05:01 and peptide, irrespective of whether the NK cell was KIR2DS4 Recognizes Complexes of HLA-C*05:01 and a educated or not. Such indifference to education fits with the ca- Nonamer Peptide pacity of KIR2DS4 to activate NK cells without the assistance of Sim et al. (1) purified and denatured HLA-C*05:01 to release signals coming from other ligand receptor interactions. the bound peptides, which were then separated and sequenced. As the peptides, mostly nonamers, arise from intracellular deg- KIR2DS4 Recognizes a Conserved Bacterial Peptide Bound radation of human proteins, they are called “self” peptides to to HLA-C*05:01 distinguish them from peptides derived from microbial patho- Having shown P2-AW was their best peptide antigen for binding gens. Aliquots of a cell line deficient in peptide loading were in- to HLAC*05:01 and engaging KIR2DS4, Sim et al. (1) searched dividually incubated with one of 46 synthetic peptides among the peptides eluted from HLA-C:05:01 for ones having corresponding to peptides isolated from HLA-C*05:01. In each tryptophan at position 8. Twelve were found, synthesized, and aliquot, HLA-C*05:01 assembled with a different peptide, tested for interaction with KIR2DS4. Two formed complexes with whereupon the complexes were transported to the cell surface HLA-C*05:01 that were recognized by KIR2DS4. The interaction and could be tested for binding to KIR2DS4-Fc. None of the with peptide MSDVQIHWF from the TM9SF4 cargo was 46 complexes bound to KIR2DS4. However, by mutating residues weak, whereas SNDDKNAWF from E3 ubiquitin ligase conferred a 7 and 8 of IIDKSGSTV, a self peptide isolated from HLA-C*05:01, strong interaction with KIR2DS4, comparable to that of P2-AW. Sim et al. (1) obtained strong binding of KIR2DS4-Fc to the P2-AY Sim et al. (1) also searched prokaryote genomes for sequences mutant peptide, in which alanine 7 and tyrosine 8 replace serine like P2-AW. Striking homology was found with the recombinase A 7 and threonine 8 of the natural peptide. Extending the analysis to protein of Helicobacter fenneliae. Positions 283 to 291 are iden- a total of 61 peptides, the only other peptides to bind HLA- tical to the first 8 residues of P2-AW, and the carboxyterminal C*05:01 and engage KIR2DS4 were variants of P2-AY in which residue has a conservative substitution. Further analysis concen- tyrosine 8 was replaced by another aromatic residue: tryptophan trated on bacterial recombinases having an acidic residue at po- in mutant P2-AW and phenylalanine in mutant P2-AF. The sition 286, the anchor residue needed for a peptide to bind HLA- strength of the interaction increased with the size of the side chain C*05:01. This selection identified 14 candidate peptides, which at position 8: tryptophan> tyrosine> phenylalanine. were made and tested for interaction with HLA-C*05:01 and To assess the functional effect of KIR2DS4 interaction with KIR2DS4. The 6 peptides that formed complexes with HLA- HLA-C*05:01, NK cells were isolated from human blood and in- C*05:01 have C-terminal phenylalanine, whereas the 8 peptides cubated with target cells expressing C*05:01 loaded with the P2- that did not form complexes have C-terminal tyrosine. The 6 dif- AW, P2-AY, or P2-AF peptide. Using flow cytometry, the NK cells ferent complexes of HLA-C*05:01 and bacterial peptide have were assayed for degranulation, a measure of NK cell activation variable affinity for KIR2DS4. By defining residues that correlate and its capacity to kill a target cell. Degranulation was observed with high or low binding to KIR2DS4, and comparing bacterial for the subset of NK cells that express KIR2DS4, but not for the recombinase sequences, Sim et al. identify >100 bacterial species many NK cell subsets that lack KIR2DS4. Other measures of NK for which antigen processing in human cells should give a peptide cell activation, such as cytokine production, correlate with the that binds HLA-C*05:01 and is recognized by KIR2DS4. extent of degranulation (1). A most unexpected observation is that The work of Sim et al. (1) on KIR2DS4 and NK cell recognition interaction between peptide-loaded HLA-C*05:01 and KIR2DS4 of bacterial antigens is complemented by that of Naiyer et al. (8) is, by itself, sufficient to activate NK cells and trigger degranula- on viral antigens. They showed that HLA-C*01:02 presents a tion. Previously, the general observation was that 2 or more dif- conserved peptide of flaviviruses, such as the Hepatitis C, Den- ferent pairs of target cell ligand and NK cell receptor are required gue, Yellow Fever, Zika, and Japanese encephalitis viruses, and to activate an NK cell (7). the complex activates NK cells that express KIR2DS2. Comparison

2of3 | www.pnas.org/cgi/doi/10.1073/pnas.1907937116 Parham Downloaded by guest on October 1, 2021 of 63 flavivirus sequences predicts that 61 of the viruses can give applies to modern humans. KIR2DS4 protects pregnant women rise to a complex of viral peptide and HLA-C*01:02 that is a ligand from preeclampsia, a disorder of poor placentation (10). Implied by for the KIR2DS2 of NK cells. Sim et al. (1) is that KIR2DS4 protects against infection by a world of bacterial pathogens. Population Genetics of KIR2DS4 Disadvantages also come with KIR2DS4, including promotion Although humans and chimpanzees are closely related, KIR2DS4 of HIV pathogenesis (11). Consequently, all human populations is the only lineage III KIR they share (Fig. 1). In separating from have 2 high-frequency KIR2DS4 alleles: one functional and one their common ancestor, the human ancestors experienced a severe not (12). In human populations, Sim et al. (1) find that the fre- population bottleneck that retained only 2 lineage III KIR. One quencies of functional KIR2DS4 and its HLA-C*05:01 ligand are was KIR2DS4, and the other was an inhibitory C1 receptor that inversely correlated. This balance limits the frequency of individ- gave rise to all modern, human-specific lineage III KIR, before uals who have both KIR2DS4 and HLA-C*05:01 and can use them becoming the nonfunctional pseudogene KIR2DP1 (9). Thus, to respond to bacteria. Such balance can arise because the KIR KIR2DS4 is 5 million years older than any other functional human and HLA loci segregate on different , and its likely lineage III KIR. This history points to KIR2DS4 having valuable cause is competing selection pressures imposed by immunity functions that contributed to early human survival and still and reproduction.

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