Arnold van der Meer

Activation of NK cells by an endocytosed receptor for soluble HLA-G.

Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy.

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.

Liver grafts contain a unique subset of natural killer cells that are transferred into the recipient after liver transplantation

In contrast to other solid organ transplantations, liver grafts have tolerogenic properties. Animal models indicate that donor leukocytes transferred into the recipient after liver transplantation (LTX) play a relevant role in this tolerogenic phenomenon. However, the specific donor cell types involved in modulation of the recipient alloresponse are not yet defined. We hypothesized that this unique property of liver grafts may be related to their high content of organ‐specific natural killer (NK) and CD56+ T cells. Here, we show that a high proportion of hepatic NK cells that detach from human liver grafts during pretransplant perfusion belong to the CD56bright subset, and are in an activated state (CD69+). Liver NK cells contained perforin and granzymes, exerted stronger cytotoxicity against K562 target cells when compared with blood NK cells, and secreted interferon‐γ, but no interleukin‐10 or T helper 2 cytokines, upon stimulation with monokines. Interestingly, whereas the CD56bright subset is classically considered as noncytolytic, liver CD56bright NK cells showed a high content of cytolytic molecules and degranulated in response to K562 cells. After LTX, but not after renal transplantation, significant numbers of donor CD56dim NK and CD56+ T cells were detected in the recipient circulation for approximately 2 weeks. In conclusion, during clinical LTX, activated and highly cytotoxic NK cells of donor origin are transferred into the recipient, and a subset of them mixes with the recirculating recipient NK cell pool. The unique properties of the transferred hepatic NK cells may enable them to play a role in regulating the immunological response of the recipient against the graft and therefore contribute to liver tolerogenicity. Liver Transpl 16:895–908, 2010.

KIR2DS5 is associated with leukemia free survival after HLA identical stem cell transplantation in chronic myeloid leukemia patients.

BACKGROUND Alloreactive NK cells play a role in tumor eradication after allogeneic HLA mismatched stem cell transplantation (SCT). The effect of NK alloreactivity in HLA identical SCT is still under debate and in particular in transplantation for chronic myeloid leukemia (CML) the data are very limited and with conflicting outcome. The aim of our study was to evaluate the effect of KIR genes and KIR ligands on leukemia free survival (LFS) and relapse rate in a well-defined, homogeneous group of CML patients phase upon HLA identical sibling SCT. METHODOLOGY We retrospectively analyzed the effect of KIRs and KIR ligands (C1 and C2) on LFS and relapse in 70 CML patients in 1st chronic phase, who had received an HLA identical sibling graft. For KIR typing we used a single PCR based KIR typing protocol that also included primers allowing for the identification of the KIR binding site on HLA-Cw (AA 77 and 80). PRINCIPAL FINDINGS The data show clear differences in transplant outcome between patients having both ligands (C1 and C2) as compared to patients having only one ligand (C1 or C2). In the latter group, the stimulatory KIR2DS5 gene was associated with improved leukemia free survival (p=0.007; hazard ratio 4.3; 95% confidence interval 1.3-6.7) and lower relapse rates (p=0.028; HR 4.3, 95% CI 1.1-9.1). In contrast, in patients carrying both ligands, KIR2DS5 was associated with reduced LFS (p=0.0056; HR 0.3; 95% CI 0.1-0.7) and higher relapse rate (p=0.02; HR 0.35, 95% CI 0.1-0.8). CONCLUSIONS Our data indicate a role for an NK mediated anti-CML response after HLA identical sibling SCT that is influenced by KIR ligands and, more importantly, by stimulatory KIRs present in the donor.

Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool for distinguishing permissible from non-permissible class I mismatches in T-cell-depleted allogeneic bone marrow transplantation

Matching for HLA has been the gold standard in bone marrow donor selection. But, with the ever increasing number of identified HLA alleles, it is becoming more difficult to find a fully HLA‐identical donor other than a sibling. Retrospective analysis revealed that HLA mismatches do not necessarily give rise to acute graft‐versus‐host‐disease (GVHD). However, we have no means of defining these ‘permissible’ mismatches before bone marrow transplantation (BMT). Thus, we set out to establish whether functional matching by means of helper and cytotoxic T‐lymphocyte precursor frequency analysis (HTLp‐f and CTLp‐f respectively) can be applied to this end. Fifty‐five recipient–donor pairs other than HLA‐identical siblings, the recipient of which received a T‐cell‐depleted graft, were analysed by high‐resolution HLA typing and/or HTLp‐f/CTLp‐f analysis. The predictive value of the CTLp‐f assay for development of acute GVHD was confirmed. More importantly, our data indicate that the CTLp‐f assay was able to discriminate permissible from non‐permissible HLA‐A, ‐B or ‐Cw mismatches, but not for DRB/DQB mismatches. The absolute number of alloreactive CTLs present in the graft correlated with the risk of acute GVHD. Although HTLp‐f and CTLp‐f together had a high negative predictive value, HTLp‐f outcome by itself was not correlated with acute GVHD. As we have no evidence yet that HTLp‐f or CTLp‐f can identify permissible DRB/DQB mismatches, high‐resolution matching for these antigens remains the best option. The combination of high‐resolution DRB/DQB typing and the CTLp‐f assay would enable the accurate prediction of the risk of acute GVHD while extending the pool of potential donors. Furthermore, it would enable adjustment of the number of T‐ cells in the graft accordingly to improve clinical outcome.

Defining early human NK cell developmental stages in primary and secondary lymphoid tissues.

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.

Interferon-γ-based mixed lymphocyte culture as a selection tool for allogeneic bone marrow donors other than identical siblings

Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy‐to‐perform cellular assays with high discriminative power. We tested a cytokine‐based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T‐lymphocyte precursor (HTLp)‐f and cytotoxic T‐lymphocyte precursor (CTLp)‐f assays. Interferon (IFN)γ was selected as a marker cytokine for (deleterious) T‐helper 1 like responses and 36 (potential) BMT donor–recipient pairs were analysed. The IFNγ‐MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp‐f, exemplified by the fact that although high IFNγ levels were observed in the class II mismatched group, certain DRB3, DQB1‐subtype and DRB1‐subtype mismatches did not give rise to IFNγ production. This might be of relevance for the detection of so‐called permissible mismatches. With regard to prediction of acute graft‐versus‐host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNγ coincided with severe aGVHD, whereas low levels were largely associated with grades 0–I. However, in the case of isolated class I mismatches the test had no predictive value. The cell‐saving IFNγ‐MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNγ‐MLC and CTLp‐f assays seems most promising for donor selection, other than identical siblings.

The proportion of follicular fluid CD16+CD56DIM NK cells is increased in IVF patients with idiopathic infertility.

One-fifth of all in-vitro fertilization (IVF) patients suffer from idiopathic infertility. A low fertilization rate is one of the most characteristic features of IVF in this group, probably caused by oocyte dysfunction. We speculate that an altered lymphocyte profile in follicular fluid (FF) may affect oocyte function and thus play a role in idiopathic infertility. Therefore, we compared levels of lymphocyte populations present in FF of 11 patients with idiopathic infertility (study group) with 29 patients in the control group, i.e. severe male factor infertility (n=17) or tubal factor infertility (n=12). Triple color flow cytometry was used to discriminate between T cells and NK cell subpopulations. In the idiopathic infertility group, a shift from T to NK cells was observed in FF as compared to the control group, caused mainly by a significant higher level of NK cells--20.3 and 13.6% (P<0.05), respectively. This high level of NK cells was due to a rise of the CD16+CD56dim NK cell subset. In peripheral blood, the NK cell levels showed a similar although not significant trend (P=0.08). As the CD16+CD56dim NK cell subpopulation is known for its cytotoxic properties, this subpopulation may negatively affect folliculogenesis and oocyte maturation, reflected by a diminished fertilization rate in the idiopathic infertility group. An altered lymphocyte profile in FF could therefore influence fertility in these patients.

Successful Transfer of Umbilical Cord Blood CD34+ Hematopoietic Stem and Progenitor-derived NK Cells in Older Acute Myeloid Leukemia Patients

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units. Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months. Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential “off-the-shelf” translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107–18. ©2017 AACR.

T cells expressing the activating NK-cell receptors KIR2DS4, NKG2C and NKG2D are elevated in paroxysmal nocturnal hemoglobinuria and cytotoxic toward hematopoietic progenitor cell lines

OBJECTIVE To investigate the presence of T cells with natural killer cell receptors (NKR) in paroxysmal nocturnal hemoglobinuria (PNH), and their potential involvement in clonal expansion of glycosylphosphatidylinositol (GPI)-deficient hematopoietic stem cells by selective immune attack to normal and not GPI-deficient hematopoietic stem cells. MATERIALS AND METHODS By flow cytometry, the frequency and number of T cells expressing NKR was evaluated in 39 PNH patients and compared to healthy controls. Elevated T cell subsets in PNH were assessed for differential cytotoxic lysis of GPI(+) and GPI(-) CD34(+) hematopoietic progenitor cell lines. RESULTS In PNH patients, the frequency (p < 0.001) and absolute number of T cells expressing the NKR CD56 (p = 0.01) were significantly increased. Furthermore, a higher percentage of T cells expressed the activating NKR NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01). Further characterization showed that these populations predominantly consist of CD8(+) effector memory CD45RA(+) T cells (T(EMRA)). NKR(+) cytotoxic T-lymphocyte lines isolated from PNH patient peripheral blood and bone marrow displayed high cytotoxicity towards CD34(+) hematopoietic progenitor cell lines and K562 cells, suggesting major histocompatibility complex class I-independent cytotoxicity. These cytotoxic T lymphocyte (CTL) lines are capable of differential lysis of GPI(+) and GPI(-) hematopoietic cell lines, however, not in all cases. This suggests that multiple factors, such as the highly activated status of in vitro cultured CTLs, influence whether GPI-dependent lysis occurs. CONCLUSIONS The increased frequency of CD8(+) effector-memory T cells with activating NKR and cytotoxicity toward hematopoietic cell lines suggests involvement in bone marrow failure and clonal expansion in PNH.