Sumati Rajagopalan

Understanding how combinations of HLA and KIR genes influence disease

Combinations of HLA and killer cell immunoglobulin–like receptor (KIR) genes have been associated with diseases as diverse as autoimmunity, viral infections, reproductive failure, and now cancer. Much as early observations of disease associations with HLA polymorphism preceded a detailed knowledge of HLA recognition by T cell receptors, the recently reported disease associations with HLA–KIR gene combinations beg for a better understanding of the underlying mechanisms.

Stress signals activate natural killer cells.

The mechanisms that regulate NK cell function as a first line of defense against infection and transformation have exceeded our expectations in terms of their sophistication and complexity. Quiescent, circulating NK cells can be activated by either soluble mediators, such as interferons and

Membrane anchoring of calnexin facilitates its interaction with its targets.

Calnexin, a chaperone that resides in the endoplasmic reticulum, participates in the quality control function of this compartment. Many glycoproteins in the process of folding associate transiently with this chaperone via interactions involving the recognition of their mono-glucosylated glycans. Some misfolded proteins which are retained in the endoplasmic reticulum exhibit prolonged association with calnexin. We have examined whether the transmembrane and cytoplasmic domains of calnexin influence the association of this chaperone with its targets. Interactions of wild type and truncated calnexin with a glycoprotein that is retained in the endoplasmic reticulum (the lymphocyte tyrosine kinase, Ltk), with membrane IgM heavy chains, and with the MHC class I heavy chain protein were investigated. A soluble calnexin molecule lacking the transmembrane domain and cytoplasmic tail does not associate with any of these proteins. When a heterologous transmembrane domain is fused to the lumenal portion of calnexin, this membrane-bound protein can bind Ltk, IgM heavy chains, and MHC class I heavy chain proteins. These results suggest that calnexin must be membrane-anchored in order to recognize its substrates.

Antagonizing inhibition gets NK cells going

Natural killer (NK) cells are key players in the innate immune system that provide rapid responses through cytokine secretion and direct lysis of stressed, infected, or transformed target cells (1). This capacity to unleash a powerful response is kept in check by inhibitory receptors such as the killer cell Ig-like receptor (KIR) family on human NK cells that recognize major histocompatibility complex class I (MHC-I) ligands on most somatic cells (2). The importance of interactions between MHC-I and KIR loci is underscored by associations of human leukocyte antigen (HLA)–KIR compound genotypes with infectious diseases, cancer, and other disorders (3). Inhibitory signaling by KIR results in a complete and proximal block of NK activation signals through recruitment of tyrosine phosphatase SHP-1 to the immunoreceptor tyrosine-based inhibition motifs (ITIM) in their cytoplasmic tail (4). Recognition of MHC-I by KIR and the resulting inhibition is influenced by the amino acid sequence of the peptide bound to MHC-I (5). In PNAS, Fadda et al. (6) now show that NK inhibition by peptide-MHC (pMHC) complexes on target cells can be overcome by specific peptide sequence variants that function as antagonists (6).

Comment on “KIR2DL4 Does not Mediate NK Cell IFN-γResponses to Soluble HLA-G Preparations”

Previous studies on the killer cell Ig-like receptor 2DL4 (KIR2DL4, CD158d) have shown that this unusual receptor is not detectable at the cell surface, and resides in endosomes of resting NK cells (1). Although direct in vitro binding of HLA-G to KIR2DL4 has not been demonstrated, soluble HLA-G accumulates in KIR2DL4-positive endosomes in NK cells and transfected cells (1). Soluble agonists of KIR2DL4 trigger an endosomal signaling pathway involving DNA damage response signaling, p21 expression and NF-κB activation to promote senescence in primary NK cells (2, 3). A physiological consequence of this signaling pathway is a senescence-associated secretory phenotype (SASP), involving proinflammatory and proangiogenic factors (including IFN-γ, IL-1β, IL-6, IL-8), that promotes tissue remodeling and angiogenesis (3). As soluble HLA-G is secreted by fetal trophoblast cells in early pregnancy, activation of NK cells in response to soluble HLA-G may contribute to remodeling of the maternal vasculature. n n nWe read with interest the article by Le Page et al. (4), who did not find evidence of functional interaction between soluble HLA-G and KIR2DL4, and question the validity of earlier reports. The conclusion that their findings “raise serious doubts about many published results suggesting that IFN-γ production by NK cells can be directly stimulated through KIR2DL4 by sHLA-G or soluble Abs” is premature, given that their work differed from other studies in several important ways: n n nThe response of NK cells to soluble HLA-G, as measured by IFN-γ, was shown to be dependent on contaminating DCs in NK cells purified using the Rosette Sep technique (Stem Cell Technologies). More highly purified NK cells using the EasySep method (Stem Cell Technologies) did not respond (4). Earlier studies on KIR2DL4 have used NK cells purified with the EasySep method (3) or the similar MACS NK negative isolation kit (Miltenyi Biotech) (1, 2). n n nContamination by bacterial products in the soluble HLA-G prepared after expression in E. coli was proposed as the basis for the stimulation of DC, which in turn activated NK cells to secrete IFN-γ (4). To avoid this problem, previous functional studies of resting NK cells used soluble HLA-G expressed by mammalian cell lines, and affinity purified with mAb W6/32 in order to enrich for properly folded HLA-G (1–3). The presence of multiple forms (β2m, free heavy chain, monomer, dimer, and high m.w. forms) of HLA-G in the bacterial preparation used by Le Page et al. (4) may have contributed to the failure to detect an NK cell response. n n nNK cells produced IFN-γ in response to “unpurified” anti-KIR2DL4 mAb #33, but not to commercial LEAF (low endotoxin azide free) mAb #33 (BioLegend) (4). In our studies (1–3), we used our own purified preparations of mAb #33. To avoid the harsh acid elution from protein A affinity columns, we purified mAb #33 by ion exchange chromatography and size exclusion (1). n n nHLA-G tetramers bound only to the contaminating DC (presumably through receptors ILT2 and ILT4) and not to resting NK cells (4). However, KIR2DL4 is not readily detectable at the cell surface, even when using Abs. KIR2DL4 is detectable in endosomes of NK cells after incubation with soluble Ab. In addition, colocalization of soluble HLA-G with KIR2DL4 in endosomes of transfected cells is KIR2DL4-dependent (1). n n n n n n nIn summary, Le Page et al. have shown that the combination of partially purified NK cells with soluble HLA-G derived from bacteria results in nonspecific and indirect activation of NK cells (4). Their study does not invalidate previous work carried out with purified NK cells and with properly folded, soluble HLA-G isolated from transfected mammalian cells.