Kathrin Schönberg

Analyses of HLA-C–specific KIR repertoires in donors with group A and B haplotypes suggest a ligand-instructed model of NK cell receptor acquisition

To determine the influence of KIR and HLA class I polymorphism on human NK cell repertoires, 32 different clonotypes representing all possible combinations of 4 inhibitory KIR and NKG2A were analyzed by multicolor flow cytometry. In donors homozygous for the common group A KIR haplotype, a significant influence of HLA-C ligands was seen: KIR repertoires were dominated by clonotypes expressing a single KIR for the respective cognate ligand, either the C1-specific KIR2DL3 or C2-specific KIR2DL1. In contrast, in donors possessing the polymorphic group B haplotypes, a similar adaptation to cognate HLA-C was lacking. We suggest that this discrepancy is largely the result of a suppressive effect of the group B-specific KIR2DL2 on the frequency of KIR2DL1(+) NK cells. In functional assays, KIR2DL2 not only recognized C1 but also C2 ligands, showing overlapping specificity with KIR2DL1. Moreover, using an NK cell differentiation assay we show sequential acquisition of KIR2DL2 before KIR2DL1 on developing NK cells. Together, these observations are compatible with a ligand-instructed model of NK cell education, in which recognition of HLA class I by an inhibitory receptor (KIR2DL2) suppresses subsequent expression of a second receptor (KIR2DL1) of related specificity. Importantly, the ligand-instructed model fits to the observed KIR repertoires in both broad KIR haplotype groups.

JAK Inhibition Impairs NK Cell Function in Myeloproliferative Neoplasms

Ruxolitinib is a small-molecule inhibitor of the JAK kinases, which has been approved for the treatment of myelofibrosis, a rare myeloproliferative neoplasm (MPN), but clinical trials are also being conducted in inflammatory-driven solid tumors. Increased infection rates have been reported in ruxolitinib-treated patients, and natural killer (NK) cells are immune effector cells known to eliminate both virus-infected and malignant cells. On this basis, we sought to compare the effects of JAK inhibition on human NK cells in a cohort of 28 MPN patients with or without ruxolitinib treatment and 24 healthy individuals. NK cell analyses included cell frequency, receptor expression, proliferation, immune synapse formation, and cytokine signaling. We found a reduction in NK cell numbers in ruxolitinib-treated patients that was linked to the appearance of clinically relevant infections. This reduction was likely due to impaired maturation of NK cells, as reflected by an increased ratio in immature to mature NK cells. Notably, the endogenous functional defect of NK cells in MPN was further aggravated by ruxolitinib treatment. In vitro data paralleled these in vivo results, showing a reduction in cytokine-induced NK cell activation. Further, reduced killing activity was associated with an impaired capacity to form lytic synapses with NK target cells. Taken together, our findings offer compelling evidence that ruxolitinib impairs NK cell function in MPN patients, offering an explanation for increased infection rates and possible long-term side effects associated with ruxolitinib treatment.

Neonatal NK-cell repertoires are functionally, but not structurally, biased toward recognition of self HLA class I.

Human natural killer (NK)-cell repertoires are biased toward more frequent expression of inhibitory killer cell Ig-like receptor (KIR) receptors for self-human leukocyte antigen (HLA) class I. Moreover, only those NK cells that express cognate receptors for self are fully functional in terms of cytotoxicity and cytokine production. It is so far unknown whether functional education and structural adaptation to HLA class I are implemented during NK-cell development and whether both processes are mechanistically connected. Here we show that NK-cell repertoires in cord blood are not yet shaped toward increased clonal frequencies of KIR for self-HLA class I as determined for the 3 major KIR ligands C1, C2, and Bw4. Nonetheless, neonatal NK cells expressing cognate KIR exhibited enhanced effector function on the level of degranulation and cytokine production. The study suggests that functional education of cognate KIR by self-HLA class I precedes structural adaptation of KIR repertoires and that both processes are not directly linked to each other.

Impaired cytotoxicity associated with defective natural killer cell differentiation in myelodysplastic syndromes

Natural killer cells are well known to mediate anti-leukemic responses in myeloid leukemia but their role in myelodysplastic syndromes is not well understood. Here, in a cohort of newly diagnosed patients (n=75), widespread structural and functional natural killer cell defects were identified. One subgroup of patients (13%) had a selective deficiency of peripheral natural killer cells (count <10/mm3 blood) with normal frequencies of T and natural killer-like T cells. Natural killer cell-deficient patients were predominantly found in high-risk subgroups and deficiency of these cells was significantly associated with poor prognosis. In the second subgroup, comprising the majority of patients (76%), natural killer cells were present but exhibited poor cytotoxicity. The defect was strongly associated with reduced levels of perforin and granzyme B. Notably, natural killer cell function and arming of cytotoxic granules could be fully reconstituted by in vitro stimulation. Further phenotypic analysis of these patients revealed an immature natural killer cell compartment that was biased towards CD56bright cells. The residual CD56dim cells exhibited a significant increase of the unlicensed NKG2A−KIR− subset and a striking reduction in complexity of the repertoire of killer cell immunoglobulin-like receptors. Taken together, these results suggest that the widespread defects in natural killer cell function occurring in patients with myelodysplastic syndromes are mostly due to either unsuccessful or inefficient generation of mature, functionally competent natural killer cells, which might contribute to disease progression through impaired immune surveillance.

Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry.

Natural killer (NK) cells provide a first line of defense against viral infections and prepare the ground for subsequent action of virus-specific T cells in a concerted way. Human NK cells use a sophisticated system of inhibitory and stimulatory receptors of the killer cell immunoglobulin-like receptor (KIR) gene family, which are expressed in a clonally distributed manner. Several studies suggest that KIR play a critical role in NK cell-mediated protection against HCV and HIV infection. As each NK cell expresses an individual set of KIR receptors that enables them to sense differences in HLA class I expression, classical measurement of NK cell function by analysis of target cell killing does not enable one to define and isolate the clinically relevant NK cell effector subsets. Here, we have developed a flow cytometry-based protocol to measure cytolytic activity together with KIR expression at a clonal level. Combined analysis of KIR expression in conjunction with cell surface mobilization of CD107 enables precise enumeration of cytolytic NK cells with defined specificity for HLA class I. Moreover, via inclusion of intracellular perforin or alternatively granzyme B, NK cells with deficient loading of cytotoxic granula can be identified. The present protocol enables identification and isolation of cytotoxic NK cells on a clonal level and enables reliable measurement in healthy as well as in pathological settings such as virus infection and hematological disease.

Get a grip on immune cells by inhibiting JAKs

ABSTRACT JAK inhibitors are approved for myelofibrosis (MF) and polycythemia vera (PV), as they reverse inflammation-associated splenomegaly and symptoms. Notably, JAK inhibitors only marginally affect disease burden. The anti-inflammatory effects of JAK inhibitors affects DC, T and NK cells explaining their therapeutic potential for limitation of cancer-associated inflammation, Graft-versus-Host Disease (GvHD) and autoimmunity.

Non-viral gene delivery into primary natural killer lymphocytes

Goffinet and Keppler reported in The FASEB Journal on the use of nucleofection for gene delivery into primary rodent lymphocytes (1). Work from us (2) is cited as evidence that “lymphocyte populations other than T cells from humans and rodents have been considered virtually refractory to nonviral gene delivery.” This notion is misleading since in this (2), as well as in several other publications from our group (3, 4), it was clearly shown that nucleofection is an effective way to transfect human natural killer cells. The transfection efficiencies observed in Trompeter et al. (2) were more than 50% for the IL-2 dependent NK cell line NK3.3 as measured by surface expression of a H-2K reporter construct and about half the efficiency for primary NK cells as measured by luciferase activity. These numbers are actually in the same range as reported by Goffinet and Keppler for rodent NK cells. In addition, it was previously shown by us and others, that nucleofection is not only suitable for transfection of activated lymphocytes, which is recommended by Goffinet and Keppler, but is also efficient in transfection of resting NK and T cells in humans and rodents, respectively (2, 5). This observation makes nucleofection an attractive tool for immunotherapeutic use of “untouched” lymphocytes, as it enables non-viral gene delivery into lymphocytes without the need for polyclonal stimulation and thus unspecific activation. REFERENCES