Reduced expansion of CD94/NKG2C+ NK cells in chronic lymphocytic leukemia and CLL-like monoclonal B-cell lymphocytosis is not related to increased human cytomegalovirus seronegativity or NKG2C deletions.

INTRODUCTION: Dysregulated NK cell-mediated immune responses contribute to tumor evasion in chronic lymphocytic leukemia (CLL), although the NK cell compartment in CLL-like monoclonal B-cell lymphocytosis (MBL) is poorly understood. In healthy individuals, human cytomegalovirus (HCMV) induces the expansion of NK cells expressing high levels of CD94/NKG2C NK cell receptor (NKR) specific for HLA-E. METHODS: We analyzed the expression of NKG2A, NKG2C, ILT2, KIR, CD161, and CD57 in 24 MBL and 37 CLL. NKG2C was genotyped in these patients and in 81 additional MBL/CLL, while NKG2C gene expression was assessed in 26 cases. In 8 CLL patients with increased lymphocytosis (≥20 × 109 /L), tumor HLA-E and HLA-G expression was evaluated. RESULTS: NKR distribution did not significantly differ between MBL and CLL patients, although they exhibited reduced NKG2C+ NK cells compared with a non-CLL group (4.6% vs 12.2%, P = .012). HCMV+ patients showed increased percentages of NKG2C+ NK cells compared with HCMV- (7.3% vs 2.9%, P = .176). Frequencies of NKG2C deletions in MBL/CLL were similar to those of the general population. Low/undetectable NKG2C expression was found among NKG2C+/- (45%) and NKG2C+/+ (12%) patients. CLL cases with increased lymphocytosis displayed especially reduced NKG2C expression (1.8% vs 8.1%, P = .029) and tumor cells with high HLA-E (>98%) and variable HLA-G expression (12.4%, range: 0.5-56.4). CLL patients with low NKG2C expression (<7%) showed shorter time to first treatment (P = .037). CONCLUSION: Reduced percentages of CD94/NKG2C+ NK cells were observed in CLL and MBL patients independently of HCMV serostatus and NKG2C zygosity, particularly in CLL patients with increased lymphocytosis, which could potentially be related to the exposure to tumor cells.

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

The Broad Spectrum of Human Natural Killer Cell Diversity.

Natural killer (NK) cells provide protection against infectious pathogens and cancer. For decades it has been appreciated that two major NK cell subsets (CD56bright and CD56dim) exist in humans and have distinct anatomical localization patterns, phenotypes, and functions in immunity. In light of this traditional NK cell dichotomy, it is now clear that the spectrum of human NK cell diversity is much broader than originally appreciated as a result of variegated surface receptor, intracellular signaling molecule, and transcription factor expression; tissue-specific imprinting; and foreign antigen exposure. The recent discoveries of tissue-resident NK cell developmental intermediates, non-NK innate lymphoid cells, and the capacity for NK cells to adapt and differentiate into long-lived memory cells has added further complexity to this field. Here we review our current understanding of the breadth and generation of human NK cell diversity.

Short-Lived Cages Restrict Protein Diffusion in the Plasma Membrane.

The plasma membrane is a heterogeneous environment characterized by anomalous diffusion and the presence of microdomains that are molecularly distinct from the bulk membrane. Using single particle tracking of the C-type lectin CD93, we have identified for the first time the transient trapping of transmembrane proteins in cage-like microdomains which restrict protein diffusion. These cages are stabilized by actin-dependent confinement regions, but are separate structures with sizes and lifespans uncorrelated to those of the underlying actin corral. These membrane cages require cholesterol for their strength and stability, with cholesterol depletion decreasing both. Despite this, cages are much larger in size and are longer lived than lipid rafts, suggesting instead that cholesterol-dependent effects on membrane fluidity or molecular packing play a role in cage formation. This diffusional compartment in the plasma membrane has characteristics of both a diffusional barrier and a membrane microdomain, with a size and lifespan intermediate between short-lived microdomains such as lipid rafts and long-lasting diffusional barriers created by the actin cytoskeleton.

Cord blood graft composition impacts the clinical outcome of allogeneic stem cell transplantation.

INTRODUCTION: Despite routine use of umbilical cord blood (CB) grafts as stem cell source for allogeneic stem cell transplantations, much remains unknown regarding their cell composition and correlation with clinical outcome. METHODS: We analyzed material from 30 CB units used for allogeneic hematopoietic stem cell transplantation by multicolor flow cytometry. Phenotypic data were correlated with various clinical outcomes such as survival, graft-versus-host disease (GVHD), relapse, rejection, viral reactivation, and bacteremia. RESULTS: We found that above-median frequencies of CD69+ T cells, naïve CD8+ T cells, and CD127+ B cells in the CB graft were each associated with significantly improved patient survival. Moreover, a statistically significant correlation was seen between higher levels of CD94+ T cells and herpes simplex virus and varicella zoster virus reactivation post transplantation. A similar correlation was seen for the frequency of CD95+ cells in total CD3+, as well as CD4+ and CD8+ T-cell subsets, and viral reactivation. Finally, a higher frequency of naïve CD8+ T cells was associated with the incidence of acute GVHD. CONCLUSION: Our study highlights the importance of further exploration of graft composition before CB transplantation as a tool for risk prediction.

Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues.

Innate lymphoid cells (ILCs) are effectors of innate immunity and regulators of tissue modeling. Recently identified ILC populations have a cytokine expression pattern that resembles that of the helper T cell subsets T(H)2, T(H)17 and T(H)22. Here we describe a distinct ILC subset similar to T(H)1 cells, which we call 'ILC1'. ILC1 cells expressed the transcription factor T-bet and responded to interleukin 12 (IL-12) by producing interferon-γ (IFN-γ). ILC1 cells were distinct from natural killer (NK) cells as they lacked perforin, granzyme B and the NK cell markers CD56, CD16 and CD94, and could develop from RORγt(+) ILC3 under the influence of IL-12. The frequency of the ILC1 subset was much higher in inflamed intestine of people with Crohn's disease, which indicated a role for these IFN-γ-producing ILC1 cells in the pathogenesis of gut mucosal inflammation.

Two complementary rat NK cell subsets, Ly49s3+ and NKR-P1B+, differ in phenotypic characteristics and responsiveness to cytokines.

Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.

CD3+/CD19+-depleted grafts in HLA-matched allogeneic peripheral blood stem cell transplantation lead to early NK cell cytolytic responses and reduced inhibitory activity of NKG2A.

Natural killer (NK) cells have an important function in the anti-tumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3(+)/CD19(+)-depleted peripheral blood stem cell (PBSC) harvests with CD34(+)-selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3(+)/CD19(+)-depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56(dim) and CD56(bright) NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3(+)/CD19(+)-depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.

GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis.

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

Natural killer cell receptor T-lymphocytes in normal and Helicobacter pylori-infected human gastric mucosa.

BACKGROUND: Helicobacter pylori infection is associated with development of chronic inflammation and infiltration of immune cells into the gastric mucosa. As unconventional T-lymphocytes expressing natural killer cell receptors are considered to play central roles in the immune response against infection, a study investigating their frequencies in normal and H. pylori-infected gastric mucosa was undertaken. MATERIALS AND METHODS: Flow cytometry was used to quantify T-cells expressing the natural killer cell markers CD161, CD56, and CD94 in freshly isolated lymphocytes from the epithelial and lamina propria layers of gastric mucosa. Thirteen H. pylori-positive and 24 H. pylori-negative individuals were studied. RESULTS: CD94(+) T-cells were the most abundant (up to 40%) natural killer receptor-positive T-cell population in epithelial and lamina propria layers of H. pylori-negative gastric mucosa. CD161(+) T-cells accounted for about one-third of all T-cells in both compartments, but the lowest proportion were of CD56(+) T-cells. Compared with H. pylori-negative mucosa, in H. pylori-infected mucosa the numbers of CD161(+) T-cells were significantly greater (p = .04) in the epithelium, whereas the numbers of CD56(+) T-cells were lower (p = .01) in the lamina propria. A minor population (< 2%) of T-cells in both mucosal layers of H. pylori-negative subjects were natural killer T-cells, and whose proportions were not significantly different (p > .05) to those in H. pylori-infected individuals. CONCLUSIONS: The predominance, heterogeneity, and distribution of natural killer cell receptor-positive T-cells at different locations within the gastric mucosa reflects a potential functional role during H. pylori infection and warrants further investigation.

Thymic stromal lymphopoietin augments the cytolytic activity of the human natural killer cell line NK-92.

Culturing the human natural killer cell line NK-92 for 24 h in the presence of thymic stromal lymphopoietin (TSLP) potentiated its cytotoxic capacity against the erythroleukemia cell line K562. Longer incubation times did not augment the NK activity any further. No synergistic effects with respect to either proliferation or cytotoxicity were observed when TSLP was mixed with suboptimal concentrations of IL-2. FACS analysis of the NK-92 cells indicated expression of TSLPR but not the other component of the TSLP receptor complex, namely IL-7Ralpha. Some of the surface molecules known to be involved in NK cell-mediated cytotoxicity were also monitored. None of the receptors analyzed altered their expression to any major extent upon culture in TSLP or IL-2. However, a limited number of NK-92 cells were observed that had a rather low CD94/NKG2A expression, which increased upon stimulation with TSLP or IL-2.

Unique characteristics of NK cells throughout the human female reproductive tract.

In this study, we have analyzed the presence and subsets of NK cells throughout the tissues of the FRT. We demonstrate that there are NK cells in the various FRT tissues and that their phenotype and regulation are largely dependent upon the FRT tissue where they reside. NK cells in the Fallopian tube, endometrium, cervix, and ectocervix expressed CD9 while blood NK cells did not. We have also found that unique subsets of NK cells are in specific locations of the FRT. The NK cells in the lower reproductive tract did not express CD94, but they did express CD16. In contrast, NK cells in the upper FRT express high amounts of CD94 and CD69, but few NK cells expressed CD16. All of these FRT NK cells were able to produce IFN-gamma upon stimulation with cytokines. Furthermore, the number of NK cells varied with the menstrual cycle in the endometrium but not in the cervix or ectocervix. These data suggest that unique characteristics of the tissues may account of specific localization of different NK cell subsets.

Reconstitution of natural killer cell receptor repertoires after unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation: analyses of CD94:NKG2A and killer immunoglobulin-like receptor expression and their associations with clinical outcome.

The effect of natural killer (NK) cell alloreactivity on the outcome of haploidentical hematopoietic stem cell transplantation (HSCT), with or without in vitro T cell depletion, remains controversial. Killer immunoglobulin-like receptors (KIRs) recognize human leukocyte antigen C and B epitopes on target cells, thereby regulating NK cell activity. To examine the recovery of CD94:NKG2A and KIR (CD158a, CD158b, and CD158e) expression by NK cells, we used flow cytometry to evaluate samples from 24 patients and their donors before and in the year following unmanipulated HLA-haploidentical/mismatched blood and marrow transplantation. Linear regression analysis demonstrated that NKG2A recovery was inversely correlated with CD158b recovery in the year following transplant. The doses of T cell subgroups CD4+ and CD8+ were inversely associated with CD158a and CD158e expression during the 2 months following transplantation. Moreover, patients with grades II-IV acute graft-versus-host disease (aGVHD) or who received "high" doses of T cells (>1.37 x 10(8)/kg) showed delayed recovery of KIRs during the 2 months following transplantation. Univariate analysis showed that patients with high CD94 expression by day 60 (>90%) or who received donors with high CD94 expression (>80%) were associated with higher transplantation-related mortality (P = .006 or .067, respectively) and poorer leukemia-free survival (P = .012 or .094, respectively). Thus, the occurrence of aGVHD or the receipt of high doses of T cells in the allograft altered KIR reconstitution. Furthermore, high levels of CD94 expression in donors or in recipients by day 60 might be a good predictor for poor prognosis.

Impaired natural killer cell lysis in breast cancer patients with high levels of psychological stress is associated with altered expression of killer immunoglobin-like receptors.

BACKGROUND: We previously reported that cancer-related psychological stress is associated with reduced natural killer (NK) cell lysis. We hypothesized that reduced NK cell cytotoxicity in patients with increased levels of stress would correlate with alterations in the expression of inhibitory NK cell receptors (killer immunoglobulin-like receptors, or KIRs). The specific aim of this study was to examine KIR expression in patients with high or low levels of psychologic stress and correlate alterations in KIR expression with NK cell function. MATERIALS AND METHODS: Two hundred twenty-seven patients underwent baseline evaluation of cancer-related psychological stress and were randomized to psychosocial intervention versus observation. From this population, two groups were defined based on pretreatment measurements of NK lytic activity, stress levels, and the availability of cryopreserved peripheral blood mononuclear cells (PBMC). Group I (n=9) had low stress by the Impact of Events Scale (IES), and high NK cell lysis at the 50:1 effector: target ratio (NK(50)=52-89%). Group II (n=8) had high stress and low NK(50) (27-52%). Lymphokine activated killer (LAK) activity, antibody dependent cellular cytotoxicity (ADCC), and expression of cytokine receptors, adhesion molecules, and killer immunoglobulin-like receptors (KIRs) were assessed in PBMC. RESULTS: Incubation of PBMC with NK-stimulatory cytokines (IL-2, IL-12, or IL-15) led to significant increases in cytotoxic activity regardless of IES/NK(50) scores. There were no significant group differences in NK cell surface expression of the IL-2 receptor components CD25 and CD122, antibody-dependent lysis of HER2/neu-positive SKBr3 cells treated with an anti-HER2/neu monoclonal antibody, expression of adhesion molecules (CD2, CD11a, CD18) and markers of activation (CD69), or expression of the KIRs CD158a, NKG2a, NKB1, and CD161. However, levels of CD158b were significantly higher in Group I after incubation in media alone or with IL-2, and CD94 expression was significantly lower in Group I after incubation with IL-2. CONCLUSIONS: In this study of a small subset of breast cancer patients chosen from a previous clinical trial of psychosocial intervention for breast cancer, impaired NK lysis in breast cancer patients with high levels of psychological stress was associated with alterations in surface expression of killer immunoglobulin-like receptors. However, immune effectors retained the ability to lyse antibody-coated targets and to initiate lymphokine-activated killer activity, irrespective of stress levels or baseline NK(50).

A high-efficiency system of natural killer cell cloning.

The culture of human natural killer (NK) cell clones has traditionally been a long, laborious process with an efficiency of only 1-2%. Recently, a stem cell growth medium (SCGM) has been described to expand preferentially polyclonal NK cells from peripheral blood. We have tested SCGM in a single cell sorting system and shown a 4-5 fold increase in the number of proliferating NK clones compared to standard RPMI media. The cloning efficiency was further enhanced by the provision of irradiated feeder cells derived from multiple donors combined with the addition of the anti-CD3 antibody, OKT3. The combination of SCGM, single cell sorting and these multiple optimisations enhanced NK cloning efficiency by more than tenfold to greater than 20% for short-term cultures when deriving 10(5) cells and as high as 10% for longer term cultures when deriving more than 2 x 10(6) cells. This novel system thus facilitates the generation of NK clones and allows larger scale studies of NK function that were beyond the scope of previous methodology.