Donor NK and T Cells in the Periphery of Lung Transplant Recipients Contain High Frequencies of Killer Cell Immunoglobulin-Like Receptor-Positive Subsets

IntroductionFor end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs. recipient NK cells.MethodsPeripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry.ResultsWithin the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR+ T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells.ConclusionHigher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx.

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Prognostic significance of natural killer cell-associated markers in gastric cancer: quantitative analysis using multiplex immunohistochemistry

Journal of Translational Medicine ◽

10.1186/s12967-021-03203-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Hee Young Na ◽

Yujun Park ◽

Soo Kyung Nam ◽

Jiwon Koh ◽

Yoonjin Kwak ◽

...

Keyword(s):

Gastric Cancer ◽

T Cells ◽

B Cells ◽

Nk Cells ◽

Natural Killer ◽

Immune Cells ◽

Nk Cell ◽

Critical Role ◽

Killer Cell ◽

Prognostic Significance

Abstract Background Natural killer (NK) cells mediate the anti-tumoral immune response as an important component of innate immunity. The aim of this study was to investigate the prognostic significance and functional implication of NK cell-associated surface receptors in gastric cancer (GC) by using multiplex immunohistochemistry (mIHC). Methods We performed an mIHC on tissue microarray slides, including 55 GC tissue samples. A total of 11 antibodies including CD57, NKG2A, CD16, HLA-E, CD3, CD20, CD45, CD68, CK, SMA, and ki-67 were used. CD45 + CD3-CD57 + cells were considered as CD57 + NK cells. Results Among CD45 + immune cells, the proportion of CD57 + NK cell was the lowest (3.8%), whereas that of CD57 + and CD57- T cells (65.5%) was the highest, followed by macrophages (25.4%), and B cells (5.3%). CD57 + NK cells constituted 20% of CD45 + CD57 + immune cells while the remaining 80% were CD57 + T cells. The expression of HLA-E in tumor cells correlated with that in tumoral T cells, B cells, and macrophages, but not CD57 + NK cells. The higher density of tumoral CD57 + NK cells and tumoral CD57 + NKG2A + NK cells was associated with inferior survival. Conclusions Although the number of CD57 + NK cells was lower than that of other immune cells, CD57 + NK cells and CD57 + NKG2A + NK cells were significantly associated with poor outcomes, suggesting that NK cell subsets play a critical role in GC progression. NK cells and their inhibitory receptor, NKG2A, may be potential targets in GC.

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151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.151 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A159-A159

Author(s):

Michael Whang ◽

Ming-Hong Xie ◽

Kate Jamboretz ◽

Hadia Lemar ◽

Chao Guo ◽

...

Keyword(s):

T Cells ◽

Cell Therapy ◽

Nk Cells ◽

Peripheral Blood ◽

Nk Cell ◽

Adoptive Cell Therapy ◽

Therapeutic Window ◽

Target Cells ◽

Healthy Donors ◽

Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

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Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

Journal of Experimental Medicine ◽

10.1084/jem.178.3.961 ◽

1993 ◽

Vol 178 (3) ◽

pp. 961-969 ◽

Cited By ~ 57

Author(s):

M S Malnati ◽

P Lusso ◽

E Ciccone ◽

A Moretta ◽

L Moretta ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Target Cell ◽

Nk Cell ◽

Class I ◽

Target Cells ◽

Cell Recognition ◽

Infected Cells ◽

Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

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Enhanced adaptive immune responses in lung adenocarcinoma through natural killer cell stimulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904253116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17460-17469 ◽

Cited By ~ 12

Author(s):

Leah Schmidt ◽

Banu Eskiocak ◽

Ryan Kohn ◽

Celeste Dang ◽

Nikhil S. Joshi ◽

...

Keyword(s):

T Cells ◽

Lung Adenocarcinoma ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Production Capacity ◽

Dependent Manner ◽

Cell Stimulation ◽

Established Disease

Natural killer (NK) cells inhibit tumor development in mouse models and their presence in tumors correlates with patient survival. However, tumor-associated NK cells become dysfunctional; thus, stimulation of NK cells in cancer is emerging as an attractive immunotherapeutic strategy. In a mouse model of lung adenocarcinoma, NK cells localized to tumor stroma with immature phenotypes and low functional capacity. To test their responsiveness within established disease, we engineered a system for inducible expression of activating ligands in tumors. After stimulation, NK cells localized inside tumors, with increased cytokine production capacity. Strikingly, T cells were also recruited to tumors in an NK cell-dependent manner, and exhibited higher functionality. In neoantigen-expressing tumors, NK cell stimulation enhanced the number and function of tumor-specific T cells and, in long-term settings, reduced tumor growth. Thus, even in established disease NK cells can be activated to contribute to antitumor immunity, supporting their potential as an important target in cancer immunotherapy.

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Slaying the Trojan Horse: Natural Killer Cells Exhibit Robust Anti-HIV-1 Antibody-Dependent Activation and Cytolysis against Allogeneic T Cells

Journal of Virology ◽

10.1128/jvi.02461-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 97-109 ◽

Cited By ~ 31

Author(s):

Shayarana L. Gooneratne ◽

Jonathan Richard ◽

Wen Shi Lee ◽

Andrés Finzi ◽

Stephen J. Kent ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Target Cells ◽

Dependent Manner ◽

Inhibitory Receptors ◽

Cell Responses ◽

Anti Hiv ◽

Hiv 1

ABSTRACTMany attempts to design prophylactic human immunodeficiency virus type 1 (HIV-1) vaccines have focused on the induction of neutralizing antibodies (Abs) that block infection by free virions. Despite the focus on viral particles, virus-infected cells, which can be found within mucosal secretions, are more infectious than free virus bothin vitroandin vivo. Furthermore, assessment of human transmission couples suggests infected seminal lymphocytes might be responsible for a proportion of HIV-1 transmissions. Although vaccines that induce neutralizing Abs are sought, only some broadly neutralizing Abs efficiently block cell-to-cell transmission of HIV-1. As HIV-1 vaccines need to elicit immune responses capable of controlling both free and cell-associated virus, we evaluated the potential of natural killer (NK) cells to respond in an Ab-dependent manner to allogeneic T cells bearing HIV-1 antigens. This study presents data measuring Ab-dependent anti-HIV-1 NK cell responses to primary and transformed allogeneic T-cell targets. We found that NK cells are robustly activated in an anti-HIV-1 Ab-dependent manner against allogeneic targets and that tested target cells are subject to Ab-dependent cytolysis. Furthermore, the educated KIR3DL1+NK cell subset from HLA-Bw4+individuals exhibits an activation advantage over the KIR3DL1−subset that contains both NK cells educated through other receptor/ligand combinations and uneducated NK cells. These results are intriguing and important for understanding the regulation of Ab-dependent NK cell responses and are potentially valuable for designing Ab-dependent therapies and/or vaccines.IMPORTANCENK cell-mediated anti-HIV-1 antibody-dependent functions have been associated with protection from infection and disease progression; however, their role in protecting from infection with allogeneic cells infected with HIV-1 is unknown. We found that HIV-1-specific ADCC antibodies bound to allogeneic cells infected with HIV-1 or coated with HIV-1 gp120 were capable of activating NK cells and/or trigging cytolysis of the allogeneic target cells. This suggests ADCC may be able to assist in preventing infection with cell-associated HIV-1. In order to fully utilize NK cell-mediated Ab-dependent effector functions, it might also be important that educated NK cells, which hold the highest activation potential, can become activated against targets bearing HIV-1 antigens and expressing the ligands for self-inhibitory receptors. Here, we show that with Ab-dependent stimulation, NK cells expressing inhibitory receptors can mediate robust activation against targets expressing the ligands for those receptors.

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Heterogeneous phenotypes of expression of the NKB1 natural killer cell class I receptor among individuals of different human histocompatibility leukocyte antigens types appear genetically regulated, but not linked to major histocompatibililty complex haplotype.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1817 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1817-1827 ◽

Cited By ~ 116

Author(s):

J E Gumperz ◽

N M Valiante ◽

P Parham ◽

L L Lanier ◽

D Tyan

Keyword(s):

T Cells ◽

Cell Surface ◽

Nk Cells ◽

Natural Killer ◽

Killer Cell ◽

Peripheral Blood Lymphocytes ◽

Class I ◽

Cell Surface Expression ◽

Target Cells ◽

Hla Type

Natural killer (NK) cells that express the NKB1 receptor are inhibited from killing target cells that possess human histocompatibility leukocyte antigen (HLA) B molecules bearing the Bw4 serological epitope. To investigate whether NKB1 expression is affected by HLA type, peripheral blood lymphocytes of 203 HLA-typed donors were examined. Most donors had a single population of NKB1+ cells, but some had two populations expressing different cell surface levels of NKB1, and others had no detectable NKB1+ cells. Among the donors expressing NKB1, both the relative abundance of NKB1+ NK cells and their level of cell surface expression varied substantially. The percentage of NKB1+ NK cells ranged from 0 to >75% (mean 14.7%), and the mean fluorescence of the positive population varied over three orders of magnitude. For each donor, the small percentage of T cells expressing NKB1 (usually <2%), had a pattern of expression mirroring that of the NK cells. NKB1 expression by NK and T cells remained stable over the 2-yr period that five donors were tested. Patterns of NKB1 expression were not associated with Bw4 or Bw6 serotype of the donor or with the presence of any individual HLA-A or -B antigens. Cells expressing NKB1 are often found in donors who do not possess an appropriate class I ligand, and can be absent in those who express Bw4+ HLA-B antigens. Family studies further suggested that the phenotype of NKB1 expression is inherited but not HLA linked. Whereas identical twins show matching patterns of NKB1 expression, HLA-identical siblings can differ in NKB1 expression, and conversely, HLA-disparate siblings can be similar. Thus NKB1 expression phenotypes are tightly regulated and extremely heterogeneous, but not correlated with HLA type.

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Natural Killer Cell Responses in Hepatocellular Carcinoma: Implications for Novel Immunotherapeutic Approaches

Cancers ◽

10.3390/cancers12040926 ◽

2020 ◽

Vol 12 (4) ◽

pp. 926 ◽

Cited By ~ 5

Author(s):

Stefania Mantovani ◽

Barbara Oliviero ◽

Stefania Varchetta ◽

Dalila Mele ◽

Mario U. Mondelli

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Immune Escape ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Current Status ◽

Tumor Immune Escape

Hepatocellular carcinoma (HCC) still represents a significant complication of chronic liver disease, particularly when cirrhosis ensues. Current treatment options include surgery, loco-regional procedures and chemotherapy, according to specific clinical practice guidelines. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as second-line therapy with limited and variable success. Natural killer (NK) cells are an essential component of innate immunity against cancer and changes in phenotype and function have been described in patients with HCC, who also show perturbations of NK activating receptor/ligand axes. Here we discuss the current status of NK cell treatment of HCC on the basis of existing evidence and ongoing clinical trials on adoptive transfer of autologous or allogeneic NK cells ex vivo or after activation with cytokines such as IL-15 and use of antibodies to target cell-expressed molecules to promote antibody-dependent cellular cytotoxicity (ADCC). To this end, bi-, tri- and tetra-specific killer cell engagers are being devised to improve NK cell recognition of tumor cells, circumventing tumor immune escape and efficiently targeting NK cells to tumors. Moreover, the exciting technique of chimeric antigen receptor (CAR)-engineered NK cells offers unique opportunities to create CAR-NK with multiple specificities along the experience gained with CAR-T cells with potentially less adverse effects.

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Elevated CD54 Expression Renders CD4+ T Cells Susceptible to Natural Killer Cell-Mediated Killing

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz413 ◽

2019 ◽

Vol 220 (12) ◽

pp. 1892-1903 ◽

Cited By ~ 1

Author(s):

Xi Chen ◽

Huihui Chen ◽

Zining Zhang ◽

Yajing Fu ◽

Xiaoxu Han ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Cd4 T Cells ◽

Nk Cell ◽

Killer Cell ◽

Adaptive Immune Response ◽

Phenotypic Analysis

Abstract Background Natural killer (NK) cells are an important type of effector cell in the innate immune response, and also have a role in regulation of the adaptive immune response. Several studies have indicated that NK cells may influence CD4+ T cells during HIV infection. Methods In total, 51 HIV-infected individuals and 15 healthy controls participated in this study. We performed the flow cytometry assays and real-time PCR for the phenotypic analysis and the functional assays of NK cell-mediated deletion of CD4+ T cells, phosphorylation of nuclear factor-κB (NF-κB/p65) and the intervention of metformin. Results Here we detected high CD54 expression on CD4+ T cells in HIV-infected individuals, and demonstrate that upregulated CD54 is associated with disease progression in individuals infected with HIV. We also show that CD54 expression leads to the deletion of CD4+ T cells by NK cells in vitro, and that this is modulated by NF-κB/p65 signaling. Further, we demonstrate that metformin can suppress CD54 expression on CD4+ T cells by inhibiting NF-κB/p65 phosphorylation. Conclusions Our data suggest that further studies to evaluate the potential role of metformin as adjunctive therapy to reconstitute immune function in HIV-infected individuals are warranted.

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The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.1133 ◽

1995 ◽

Vol 181 (3) ◽

pp. 1133-1144 ◽

Cited By ~ 359

Author(s):

J E Gumperz ◽

V Litwin ◽

J H Phillips ◽

L L Lanier ◽

P Parham

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Inhibitory Effect ◽

Glycosylation Site ◽

Class I ◽

Target Cells ◽

Cell Clones

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones.

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KIR Positive Subsets of Human CD56+CD3+ NKT Cells Are Different in Frequency from Equivalant KIR Positive CD56+CD3- NK Cell Subsets.

Blood ◽

10.1182/blood.v110.11.3878.3878 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3878-3878

Author(s):

Ilka Bondzio ◽

Andreas Arendt ◽

Jurgen Schmitz ◽

Volker Huppert

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Cytotoxic T Cells ◽

Killer Cell ◽

Nkt Cells ◽

Nkt Cell ◽

Cell Clones ◽

Number Of Publications

Abstract Killer cell immunoglobulin-like receptors (KIRs) are known to modulate the cytotoxic ability of human Natural Killer (NK) cells, as well as a subset of T cells. To date, only a very small number of publications have discussed the role of KIRs on T cells, e.g. CMV-specific CD4+CD28-KIR+ cytotoxic T cells (van Bergen, J., J Immunol. 2004), so we investigated whether CD56+CD3+ NKT cells might also have KIR-positive subsets. Whole human blood as well as magnetically sorted human CD56+CD3+ NKT cells were analyzed for their expression of various KIR molecules using a novel panel of fluorochrome-conjugated, anti-KIR monoclonal antibodies (CD158a/h (KIR2DL1/DS1), CD158b (KIR2DL2), CD158e (KIR3DL1), CD158i (KIR2DS4), KIR2D; Miltenyi Biotec). KIR-positive CD56+CD3+ NKT cells were identified in every donor tested. Donors possessing NK cells of a specific KIR phenotype also possessed CD56+CD3+ NKT cells with the same KIR phenotype. KIRs were also expressed in a clonal fashion on CD56+CD3+ NKT cells, similarly to NK cells. The investigated KIRs were also shown to be expressed on unseparated NK and CD56+CD3+ NKT cells from whole blood. In addition, the ratio between KIR expression on NK and CD56+CD3+ NKT cells was calculated for each donor analyzed. The results show that there is no correlation between the frequencies of KIR expression on NK cells with that of CD56+CD3+ NKT cells. For example, the expression of CD158a/h in one donor was found to be the highest of all CD56+CD3+ NKT cells analyzed, but the lowest of all NK cells by comparison to the other donors tested. For all KIR phenotypes analyzed, the frequency of KIR+ NK cells was higher than the frequency of KIR+ CD56+CD3+ NKT cells in all samples (range: 1.1 to 25.3-fold higher). Interestingly, the frequency of KIR+ NK cells versus KIR+ CD56+CD3+ NKT cells differs significantly between donors: in one donor the frequency of KIR expression is between 7.3 to 25.3-fold higher in NK cells for multiple KIR phenotypes, while this range is more narrow in other donors (2.0–5.4-fold higher). The frequencies of CD56+CD3+ NKT cell subsets staining positive for particular KIRs differ significantly between donors, e.g. for CD158b, the number of positive CD56+CD3+ NKT cells fall within a range of 4.8% to 43.3%. For CD56+CD3+ NKT cells sorted with MACS® Technology, a similarly wide-ranging distribution of CD158b (KIR2DL2) expression was found (0.85%–5.82%), though at a lower level. Further research will be required to explore these differences as they may point to different mechanisms of KIR regulation. The identification of KIR-positive CD56+CD3+ NKT cells may also provide an opportunity for their use for functional KIR studies instead of NK cell clones, as the cloning of CD56+CD3+ NKT cells may prove easier (i.e. using standard T cell cloning methods) than that of NK cells.

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