Natural Killer Receptor Expression Research Articles

Peripheral Blood (PB) CD56bright and CD56dim Do Not Exhibit Differences in NKR Expression Compared to Their Cord Blood (CB) Counterparts: CB CD56dim May Represent an Intermediary between CB CD56bright and PB CD56dim.

NK cells are characterized by absent CD3 and expression of CD56. CD56 NK cells are classified into CD56dim (90%) that are primarily cytotoxic, and CD56bright that secrete cytokines (Kaplan et al PNAS 1998; Shankaran et al Nature 2001). NK subsets carry out their respective functions based on their repertoire of NK receptors (NKRs), both activating and inhibiting (Moretta et al Annu Rev Immunol 2001). PB CD56dim cells express high concentrations of killer-cell immunoglobulin receptors (KIR) and C-type lectin receptors. PB CD56bright NK cells have increased expression of C-type lectin receptors, but have low expression of the KIRs (Nagler J. Immunol 1989). CD16 (the Fcγ receptor III or FcγRIII) which is responsible for binding to antibody-coated targets and initiating antibody-dependent cellular cytotoxicity (ADCC) has increased expression in CD56dim vs CD56bright NK cells (Leibson et al Immunity 1997). While the characterization and function of PB NK subsets and NKR expression are well described, there is little information regarding CB NK subsets and NKRs. We previously demonstrated a significant increase in CB NK NKR expression in ex-vivo engineered CB MNC following 48 hours of induction with IL-2, IL-7, IL-12, and Anti-CD3 (Ayello/Cairo et al BBMT 2006). In this study, we compared PB vs. CB NK subsets and NKR expression. PB and CB cells were positively selected for CD56+ using CD56 magnetic beads (Miltenyi, Aubrun, CA). CD56+ cells were sorted into CD3−/CD56bright and CD3−/CD56dim subsets by FACS sorter and NK receptor expression (CD16, CD158a {KIR2DL1} CD158a, h {KIR2DL1 and KIR2DS1}, CD158b {KIR2DL2}, CD161, NKG2A, NKG2C, NKG2D, Nkp44, NKp46, Becton Dickinson, Mountainview, CA,) of each subset was analyzed by flow cytometry. CD56+ selection yielded >89% purity (PB-96%, CB-89%). There was no statistical difference (mean ± SEM) in the receptor expression between the CB CD56bright and PB CD56bright and also between the CB CD56dim and PB CD56dim. However, PB CD56bright vs. PB CD56dim had increased expression of NKG2A (93.28 ± 2.83 vs. 66.64 ±7.74, p< 0.015), NKG2C (62.11 ±11.79 vs. 8.08 ± 2.98, p< 0.12), NKp44 (61.52 ±10.35 vs. 3.79 ± 2.32, p< 0.005) and NKp46 (92.53 ±3.31 vs. 64.66 ± 12.34, p< 0.05). PB CD56dim had increased expression of CD16 (93.30 ± 2.71 vs. 61.15 ± 11.79, p< 0.07) compared to PB CD56bright. The CB CD56dim compared to CB CD56bright has increased CD16 expression (85.06 ± 6.75 vs. 40.91 ± 5.74, p< 0.004) only. The PB CD56bright only differed from the CB CD56dim with respect to NKp44 (p< 0.021). The CB CD56bright differed from the PB CD56dim with respect to KIR2DL1 (p< 0.026), CD161 (p < 0.05), NKG2A (p < 0.025), and NKG2C (p < 0.05). In conclusion, there does appear to be a definite difference in NKR expression in PB CD56bright vs. PB CD56dim. However, PB vs. CB CD56dim and PB vs. CB CD56bright did not have any statistically significant NKR expression differences suggesting that the subsets have similar phenotypes in their respective cell source. Based on these results, we postulate that the CB CD56dim subset might reflect an intermediary step in development transitioning from CB CD56bright to PB CD56dim.

Reduced Natural Killer Function Accompanies MDS Disease Progression and Is Restored by Lenalidomide (CC5013, Revlimid®) Via a Mechanism Divergent from Interleukin-2 (IL-2).

Background: Patients with myelodysplastic syndromes (MDS) display several abnormalities of T cell and NK cellular immunity. Tumor lysis by NK cells occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs. Lenalidomide is an immunomodulatory (IMiD) drug structurally related to thalidomide, which has proven clinical efficacy for the treatment of low-risk MDS (List et al, NEJM 2005; 352:549). Investigations in patients with multiple myeloma (MM) reported that thalidomide was associated with enhanced cytolytic function and increased NK cells in responding patients. We investigated the action of lenalidomide on NK function and activating NK receptor expression in patients with MDS.Methods: MDS patients were categorized according to WHO category, age, sex, IPSS, and cytogenetics. Peripheral blood cells (PBMCs) were isolated from patients and normal donors and NK receptor expression determined in paired healthy and patient PBMCs by 3-color flow cytometry. NK receptor expression was analyzed for CD158a (KIR2DL1, KIR2DS1), CD158b (KIR2DL2, KIR2DL3, KIR2DL3), KIR3DL1, KIR2DS4, NKG2A, NKG2D, NKp30, NKp44, and NKp46. Cytotoxicity assays were performed using 5-hour 51Cr-release assays.Results: Forty-eight MDS patients and 37 normal donors were analyzed, demonstrating that NK cytolytic function was reduced in the patient population (19% ± 21 S.D. vs. 44% ± 21) (p<0.001). Reduced NK function in MDS was significantly associated with higher IPSS (p=0.01), abnormal karyotype (p=0.05), presence of excess blasts (p=0.01), and age-adjusted bone marrow hypercellularity (p=0.04). MDS patients had reduced display of the activating receptor NKp30, accompanied by NKG2D (an activating C lectin-like (NKG2)-family receptor) downregulation that closely correlated with impaired NK function (p=0.001). NK-mediated cytotoxicity and NKR expression were examined after treatment with lenalidomide 5μM for 72 hours or IL-2 in normal donors and MDS patients. Lenalidomide augmented NK lytic function in normal donors (vehicle, 42% ± 15 vs. lenalidomide, 71% ± 17; P≤0.01 by t test) and in 10 out of 17 (59%) of the MDS patients (vehicle, 25% ± 25 vs. lenalidomide 42% ± 33). IL-2 increased NK cytolytic function in all MDS patients (vehicle, 16% ± 16 vs. lenalidomide, 47% ± 33) and the percentage of NK cells expressing CD69 was increased from both MDS patients and normal controls. Similarly, the percentage of NK cells expressing NKp30, NKG2D, NKp44 but not NKp46 was significantly enhanced by IL-2 culture. In contrast, lenalidomide treatment failed to induce NKR expression in MDS patients and normal controls.Conclusions: These findings suggest that impairment of NK cytolytic function in MDS derives in part from reduced display of the activating NK receptors such as NKG2D, which accompanies disease progression. Lenalidomide augments NK function in some patients; however, the mechanisms of functional augmentation by IL-2 and lenalidomide are divergent. Evasion of NK immunosurveillance may be an important factor associated with MDS disease progression.

Enhanced NK Cell Activation, Cytotoxicity and Ex-Vivo Expansion (EvE) of Cryopreserved Cord Blood (CB) Natural Killer (NK) Cells: Potential Role for CB NK Cells in Adoptive Cellular Immunotherapy (ACI).

CD56+ NK subsets exhibit differential NK receptors (NKR ) such as NCR profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytoxicity receptors (NCR) involved with tumor target recognition (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several NKRs such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opinion in Immunol, 2004). Target cell killing by activated NK cells via the granule-dependent pathway is a common mechanism of NK and CTLs and degranulation is followed by the expression of lysosomal-associated membrane protein-1 [LAMP-1] on the cell surface (Penack et al, Leukemia, 2005). CB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after UCBT (Cairo, et al, Transfusion, 2005). We have demonstrated the ability to EvE CB in short-term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and EvE (CTCTE) CB with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2a (Ayello/Cairo et al BBMT, 2006). In this study, we compared short-term culture (48 hrs) with prolonged cultures (4 to 10 days) on expansion, expression of NCR, NKG2, KIR and cytolytic ability and mechanisms in CTCTE CB. Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]. NKR expression (CD94, NKG2D, Nkp44 and KIR2DS4), intracellular perforin, granzyme B activity and LAMP-1 receptor (CD107a) expression were determined by flow cytometry. Cytoxicity was measured by europium release assay and tumor targets used were K562, Daudi, neuroblastoma (SHSY5Y) and AML (Kasumi-1) at a 20:1 E:T ratio. C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4±0.43 vs 23.7±2.%, p<0.001). Significant increases were seen in activating KIR2DS4 at day 10 vs 2 in ABCY in both CD3−/16+/56+dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9± 0.2%, p<0.001, respectively). In contrast, NCR expression in CD3−/16+/56+dim NKp44 subset was significantly decreased at day 10 vs 2 of EvE CB in ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). Granzyme B expression was increased from day 2 to 10 (25.8± vs 45.1± 1.7%, p<0.001) yet perforin was decreased in EvE CB in ABCY at day 7 vs 2 (68.3±2.19 vs 84.3±1.3%, p<0.001). CD107a expression was significantly increased at day 7 vs 2 in ABCY EvE CB (12.95±1.47 vs 69.34±2.22%, p<0.001). In addition, significant increases in cytolytic activity was demonstrated at day 7 vs 2 of EvE CB cells in ABCY against tumor targets K562 (71.5±±0.81 vs 53.8±3.9%, p<0.001), Daudi (63.9±0.73 vs 31.8±1.8%, p<0.001), SYSY5Y (76.8±6.5 vs 57.5±3.4%, p<0.05) and Kasumi-1 (56.6.5±0.4 vs 38±1.1%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed at a later date, EvE and activated for up to 10 days to yield significantly increased cytotolytic activity against NHL, AML and neuroblastoma with increased expression of NK KAR KIR2DS4 and granzyme B, LAMP-1 degranulation (NK activation) but decreased NK C-lection CD94/NKG2D, NCR NKp44 and perforin expression.

T-cell type lymphoproliferative disease of granular lymphocytes (LDGL) is equipped with a phenotypic pattern typical of effector cytotoxic cells

By analyzing the expression of several cytotoxic markers, killer-immunoglobulin-like receptors (KIRs), CD94/CD159, CD314 and natural cytotoxicity receptors (NCRs), in 22 CD3+ lymphoproliferative disease of granular lymphocyte (LDGL) patients we investigated whether granular lymphocytes (GLs) displayed the phenotype of fully differentiated cytotoxic cells. Our results demonstrate that GLs express a pattern consistent with fully differentiated CTLs. KIRs are expressed only in a fraction of patients (7/22), as is CD94/CD159 (5/22). In conclusion, GLs in CD3+ LDGL patients typically show the phenotype of fully differentiated CTL, whereas the expression of NK receptors does not represent a common feature of the proliferating clone.

Tachykinins and their Receptors in Human Malignancies

The possibility of links between psychosocial factors and cancer incidence and progression has generated considerable scientific and public interest. Tachykinins, including substance P, neurokinin A and B, hemokinin-1 and endokinins, are a family of neuropeptides, acting through three types of transmembrane G-protein coupled receptors denoted NK1, NK2 and NK3. Besides their role as neurotransmitters in peripheral and central nervous system, tachykinins and their receptors are also expressed in several non neuronal cells contributing to the fine connections between nervous systems and peripheral organ system such as respiratory, cardiovascular, immune, endocrine, gastrointestinal and genitourinary. Being so much involved in regulating physiological functions, they, of course, can concur to pathological conditions including cancer. Tachykinins can act on different steps of carcinogenesis. Tumors expressing NK receptors, such as astrocytoma, glioma, neuroblastoma, pancreatic cancer and melanoma, can misuse tachykinin-induced signaling, operating in normal cells, to promote proliferation and survival of cancer cells and to release cytokines and soluble mediators favoring tumor growth. In neuroblastoma, breast and prostate carcinomas tachykinins facilitate tumor metastatic infiltration in the bone marrow. In neuroendocrine carcinoma, tachykinins are responsible of symptoms associated with these pathologies including flushing, diarrhea, wheezing and right heart disease. In addition, regardless tumor histology, tachykinins may favor cancer incidence and metastatic progression by influencing blood flux and neovascularization in tumor formation as well as inducing immunosuppression mediated by neurogenic inflammation due to stress or surgery. However, the precise involvement of tachykinins in cancer pathologies and the potentiality to become effective pharmacological drug targets remain to be fully defined.

Activation and enhancement of ex vivo (EvE) expanded cryopreserved Cord Blood (CB) Natural Killer (NK) cells, cytolytic potential, and NK receptor (NKR) expression: Potential role for CB NK in Adoptive Cellular Immunotherapy (ACI)

Activation and enhancement of ex vivo (EvE) expanded cryopreserved Cord Blood (CB) Natural Killer (NK) cells, cytolytic potential, and NK receptor (NKR) expression: Potential role for CB NK in Adoptive Cellular Immunotherapy (ACI)

Pathogenic Mechanisms of HIV Disease: The Role of Viral Replication and Immune Activation

HIV replication and the immune response to the virus lead to a state of generalized immune activation, which drives the pathogenesis of HIV disease. HIV-induced immune activation results in increased T-cell turnover (production and destruction), increased death of T cells, a decline in the size of the CD4+ T-cell pool, and a state of activation-induced immunodeficiency. In many HIVinfected individuals, these phenomena can be reversed or mitigated with effective antiretroviral therapy (ART), which blunts HIV replication and can reduce plasma levels of viremia to undetectable levels. Recent data from our laboratory have demonstrated fundamental differences between viremic and aviremic individuals with regard to the physiologic state of the resting CD4+ T-cell reservoir of HIV and the phenotype and function of T cells, B cells and natural killer (NK) cells. In terms of the CD4+ T-cell reservoir, resting cells in viremic versus aviremic individuals differentially express certain genes associated with HIV replication and are continually poised to express virus as a result of continual activation related to viremia; thus, true latency likely does not exist in these patients. We had previously reported that much greater stability exists in aviremic individuals, suggesting the presence of a truly latent reservoir of virus in this population. However, recent data indicate that the normal physiologic process of low-level immune activation in aviremic individuals sustained the low level turnover and propagation of the latent HIV reservoir. Previously, we have demonstrated that HIV-mediated hyperactivation induces the terminal differentiation of B cells. More recently, we have determined that certain B-cell genes are upregulated in HIVviremic individuals as compared with HIV-aviremic individuals. Many of these genes are associated with terminal B-cell differentiation and death by apoptosis. Recent data indicate that there is a balance between expression of survival and apoptotic genes in HIV-infected individuals that is impacted by the presence and level of viremia. In viremic individuals we also have characterized a population of aberrantly activated CD56NK cells that manifest lower-than-normal expression of Natural Cytotoxicity Receptors and a normal or increased expression of inhibitory NK receptors. This dichotomy is reflected in decreased cytotoxic function and decreased secretion of TNF-alpha and interferon-gamma. In addition, NK cells from viremic individuals express Fas on their surfaces at significantly increased levels, and are more susceptible to apoptosis upon exposure to sFASL. Furthermore, viremia impacts the NK cell-dendritic cell interactions that are critical to normal immune responsiveness. Taken together, our data indicate that HIV pathogenesis involves both direct and indirect effects of HIV and a relentless cycle of aberrant immune activation that drives the disease process in HIV-infected individuals. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Natural Killer Cells with NKDG2D Cytotoxicity but NKp30-Dull Phenotype Predominate in Myelodysplastic Syndrome (MDS).

Natural Killer (NK) function in patients with MDS as measured by non-MHC-restricted cytotoxicity and activation-dependent cell cytotoxicity (ADCC) are reduced in patients with MDS, however, the mechanisms of the functional impairment are not known. Tumor cytolysis occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs, which control signaling events that lead to polarized movement of perforin-containing granules toward the NK-tumor contact area. We found that NK cells from 23 out of 35 patients with MDS (66%) displayed reduced lysis of K562 tumor cells compared to age-matched normal controls (p<0.01). To better characterize this defect, we evaluated patient NK function against differential tumor targets including the MDS1 cell line established from an MDS patients. We found that MDS1 incited non-MHC-restricted lysis. Unactivated PBMCs, unactivated NK cells, NK cell lines (NK92 and NKL) but not purified unactivated T cells from normal donors killed MDS1 in 4-hr 51Cr-release assays. Normal NK cells and NK cell lines were also found to rapidly redistrubute perforin granules after exposure to MDS1suggesting that a perforin-dependent lytic pathway was activated. We then performed simultaneous cytolytic assays with K562, MDS1, and the 721.221 B cell lymphoma cell line as target cells. We found that NK cells from MDS patients had greater lytic activity against MDS1 (average 24% vs. average 8% at 50:1 Effector:Target ratio, respectively, p<0.01) Antibody-blocking experiments demonstrated that the NKL cell line and PBMCs from 8 out of 10 MDS patients predominantly used the NKG2D activating receptor to kill MDS1. Consistent with this finding, we showed that MDS1 cells express the major human stress-inducible endogenous proteins MICA and MICB, which are NKG2D ligands. In contast, lysis by NK92 cells and normal PBMCs was not appreciably reduced by NKG2D blocking antibodies suggesting that other unidentified NKR(s) also mediate lysis. To identify the NKRs expressed in MDS patients, we performed immunophenotyping for both the activating NKRs and inhibitory NKRs compared to age-matched normal controls. We found that two activating receptors, NKp30 and CD244 (2B4), were significantly reduced on NK cells from all MDS patients regardless of their ability to lyse NK targets. Inhibitory NKR expression and function were normal. Interestingly, NKG2D expression correlated with reduced cytolytic function. Similar to studies on normal NK cells with low NKp30 and NKp46 (NCRdull) phenotypes, these results suggest that low NKp30 expression leads to predominant NKG2D utilization for tumor cell lysis, which is reduced in MDS patients with defective NK function. Our findings provide critical information about potential importance for immunosurviellance through NKG2D-NKG2D ligands.

Up-Regulation of NK Cell Activating Receptors Associated with Elevated IL-15 Levels Post-Transplant.

NK cells express diverse activating and inhibitory receptors, which collectively determine the NK cytotoxic response. Because NK cells can be potent anti-tumor effectors in the post transplant period of minimal residual disease, we investigated the receptor expression on NK cells following non-myeloablative, HLA-matched, allogeneic hematopoietic stem cell transplant (HSCT). We focused on activating receptors, comparing the expression of natural cytotoxicity receptors (NCR) and C-type lectin receptors on circulating NK cells at one, three, six and twelve months following HSCT with that of the donor-derived mobilized stem cell apheresis. During the first post-transplant month, the absolute numbers of NK cells recovered to normal levels in the 14 patients studied, but the subpopulation of CD56++(bright) CD16− NK cells increased disproportionately compared to the more common CD56+(dull) CD16+ NK cells. By six months the proportions of the NK cell subsets normalized to pre-transplant levels. At one month post transplant, the median percentage of NK cells expressing the anti-tumor NCR NKp46 increased from 15 to 64% and that expressing NKp30 increased from 23 to 40% as compared to the donor's NK cells; expression remained elevated during the first year. Expression of NKG2D, the homodimeric activating C-type lectin receptor, similarly increased post-transplant. CD94 was also upregulated on NK cells, but the activating heterodimeric partner of CD94, NKG2C, did not change significantly. To investigate these shifts in NK populations and receptor expression, we investigated the effect of cytokine milieu post transplant on these shifts in NK populations and receptor expression. We have determined that plasma levels of IL-15, a cytokine critical in NK differentiation and expansion, increase more than 50-fold in these HSCT patients from pretreatment to the day of transplant and decline in the first weeks post transplant, inversely proportional to NK recovery. When NK cells were cultured with rIL-15, we observed a disproportionate expansion of CD56++ NK cells and a rapid up-regulation of the NCR and NKG2D receptors, similar to the changes observed post-transplant. These data suggest that the cytokine milieu of transplant, in particular elevated levels of IL-15, may contribute to anti-tumor efficacy post transplant by affecting the recovery of NK subsets and modulating expression of activating receptors.

Optimal Ex-Vivo Expansion (EvE) of Cord Blood (CB) Natural Killer (NK) Cells, NK Cytotoxicity and NK Receptor (NKR) Expression: Potential for CB Adoptive Cellular Immunotherapy (ACI).

CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several triggering receptors such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta, et al, Curr Opinion in Immunol, 2004; Marcenaro et al, Eur J Immunol, 2003). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood, 2001; Locatelli, et al Blood,1999). We demonstrated the ability to EvE CB in short term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2A (Ayello/Cairo et al, BBMT, 25a, 2004). In this study, we compared short-term cultures (48 hrs) with prolonged cultures (4 to 10 days) on expansion, maturation and expression of NCR, NKG2, KIR and cytolytic mechanisms in previously cryopreserved CB that were TCTE. CB was cryopreserved and thawed by the NHLBI/COBLT method. (Kurtzberg/Cairo, Transfuison, 2005). Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free medium alone or with anti-CD3 (50 ng/ml, IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) (ABCY). NK receptor expression (CD94, NKG2C, NKG2D, NKp44, NKp46, KIR2DS4) and intracellular perforin and granzyme B activity were determined by flow cytometry. NK and LAK cytotoxicity was measured by europium release assay. Significant increases were seen in NK activating KIR2DS4 at day 10 vs 2 in ABCY both in CD3−/16+/56+ dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9±0.2%, p<0.001, respectively). C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4 ±0.43 vs 23.7±2.0 %, p<0.001). A significant increase was seen in NK (CD3−/16+/56+dim) KIR3DL1 subset at day 10 vs 2 (38.3±2.8 vs 18.9±6.3 7%, p<0.05). In contrast, NCR expression in CD3−/16+/56+ dim NKp44 subset was significantly decreasedat day 10 vs 2 of EvE with ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). A significant decrease was seen in CD3−/16+/56+ dim NKp46 expression following day 10 vs 2 (8.5±0.2 vs 23.5±1.2 %, p<0.001). Perforin expression demonstrated a significant decrease in ABCY at day 10 vs 2 (55.7±1.8 vs 84.3±1.3%, p<0.001) yet increasing levels of granzyme B from day 2 to 10 (25.8±1.8 vs 45.1±1.7%, p<0.0001). A significant increase in CB NK and LAK cytotoxicity was seen in ABCY on day 10 vs 2 (NK: 71.5 ±1.6 vs 53.8±10.3%, p<0.001; LAK: 63.2±0.24 vs 31.8±1.8%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed and at a later date EvE and activated for 7–10 days to yield viable NK subsets. EvE in ABCY at 10 days yielded increased expression of NK KAR (CD56+dim and bright) and granzyme B expression but decreased NK C-lectin CD94/NKG2D, NCR NKp46 and NKp48 and perforin expression.

Expression of inhibitory natural killer receptors on tumor-infiltrating CD8+ T lymphocyte lineage in human endometrial carcinoma

To investigate the expression of natural killer receptors (NKRs) within the human tumor milieu, we directly examined the in vivo expressions of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human endometrial carcinoma (EC). In total, 22 patients with stage IA-IIIA EC were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique. The subpopulations of immunocytes were quantified, and expressions of NKRs on CD8+ T cells were analyzed by triple-color flow cytometry. CD8+ T cells express higher ratios of CD94 and NKG2A in TILs than in peripheral blood mononuclear cells (PBMCs) in human EC. Flow cytometry reveals that 15.90% of CD3+CD8+ TILs compared with 2.10% of CD3+CD8+ PBMCs express the NKG2A molecules (P < 0.001). The percentage expressions of CD94 are 8.40% in CD3+CD8+ TILs and 3.80% in CD3+CD8+ PBMCs (P= 0.013). The numbers of CD8+ T cells expressing CD158b and NKB1 are higher in CD3+CD8+ PBMCs in EC than in normal (CD158b: 10.70% vs 2.60%, P < 0.001; NKB1: 2.20% vs 0.40%, P= 0.018, respectively). Increased expression of CD94/NKG2A restricted to tumor-infiltrating CD8+ T cell subsets may shape the cytotoxic responses, which indicate a possible role of tumor escape from host immunity in human EC.

2B4 expression on natural killer cells increases in HIV-1 infected patients followed prospectively during highly active antiretroviral therapy.

Human immunodeficiency virus (HIV)-1 infection influences natural killer (NK) cell expression of inhibitory NK receptors and activating natural cytotoxicity receptors. It is unknown whether expression of the co-stimulatory NK cell receptor 2B4 (CD244) on NK cells and CD3+ CD8+ cells are affected by highly active antiretroviral therapy (HAART), low-level viraemia, proviral-DNA or immune activation in HIV-1 infected patients. A total of 101 HAART-treated HIV-1 infected patients with < or = 200 HIV-RNA copies/ml were followed prospectively for 24 months. HIV-RNA was investigated 3-monthly and 2B4 expression on CD3- CD16+ NK cells and CD3+ CD8+ cells, proviral-DNA and plasma soluble tumour necrosis factor receptor (sTNFr)-II were investigated 6-monthly. For comparison, 2B4 expression was investigated in 20 healthy individuals. The concentration of 2B4+ NK cells was initially reduced in HIV-1 infected patients (P < 0.001) but increased to a normal level during the 24 months' follow-up. The concentration of CD3+ CD8+ 2B4+ cells in HIV-1 infected patients was normal and did not change during follow-up. The relative fluorescence intensity (RFI) of 2B4 increased on both NK cells and CD3+ CD8+ cells during follow-up (both P < 0.001). Higher levels of proviral-DNA carrying cells and plasma sTNFrII were associated with reductions in the concentration of 2B4+ NK cells (all P < 0.05). HIV-RNA had no effect on 2B4 expression on NK cells or CD3+ CD8+ cells. These findings demonstrate that the concentration of 2B4+ NK cells normalizes during long-term HAART in HIV-1 infected patients. The finding that proviral-DNA and sTNFrII were associated negatively with the concentration of 2B4+ NK cells suggests that immune activation in HIV-1 infected patients receiving HAART influences the target cell recognition by NK cells.

Development of Murine Plasmacytoid Dendritic Cells Defined by Increased Expression of an Inhibitory NK Receptor, Ly49Q

Plasmacytoid dendritic cells (PDCs) are defined in mice by a unique combination of markers: CD11c, B220, and Ly6C/G. We have reported previously that PDCs express Ly49Q, a lectin-type killer cell inhibitory receptor. We now find that different expression levels of Ly49Q define sequential developmental stages of PDCs in bone marrow. Although PDCs in spleen and lymph nodes express high levels of Ly49Q, a significant portion of CD11c(+)B220(+) PDCs in bone marrow lack Ly49Q, as well as the CD4 and MHC II. Purified Ly49Q(-) marrow PDCs spontaneously up-regulate Ly49Q after overnight culture without cell proliferation and acquire most features of typical PDCs in spleen. When exposed to TLR ligands, such as CpG-oligodeoxynucleotide and hemagglutinating virus of Japan (Sendai virus), Ly49Q(-) PDCs increase CD86 and MHC class II expression but produce less IFN-alphabeta, IL-6, and IL-12p70 than Ly49Q(+) PDCs, although they are able to produce comparable amounts of TNF-alpha. However, interestingly, Ly49Q(-) PDCs do not produce TNF-alpha in response to the TLR2 ligand, Pam3SCK(4), whereas Ly49Q(+) PDCs did. Therefore, Ly49Q is a new marker to identify a precursor form of PDCs that participates in innate immunity.

IFN Regulatory Factor-2 Deficiency Revealed a Novel Checkpoint Critical for the Generation of Peripheral NK Cells

NK cell development is far less understood compared with that of T and B cells despite the critical importance of NK cells in innate immunity. Mice lacking the transcription factor IFN regulatory factor-2 (IRF-2) are known to exhibit NK cell deficiency. However, the role of IRF-2 in NK cell development has remained unclear. In this study we found that NK cell deficiency in the periphery in IRF-2-deficient mice was due to selective loss of mature NK cells, but not to maturation arrest, and NK cells in these mice exhibited very immature surface phenotypes (CD11b(low)Dx5(low)) with highly compromised NK receptor expression. In contrast, IRF-2-deficient NK cells in bone marrow (BM) showed relatively mature phenotypes (CD11b(low)Dx5(high)) with less compromised NK receptor repertoire. Furthermore, BM NK cells in IRF-2-deficient mice were found to proliferate almost normally, but underwent accelerated apoptosis. These observations indicated that NK cell maturation could advance up to a late, but not the final, stage in the BM, whereas these cells were incapable of contributing to the peripheral NK cell pool due to premature death in the absence of IRF-2. In contrast, NK cell numbers and Ly49 expression were much more severely reduced in BM in IL-15-deficient mice than in IRF-2(-/-) mice. The differential peripheral and central NK cell deficiencies in IRF-2(-/-) mice thus revealed a novel late checkpoint for NK cell maturation, distinct from the early IL-15-dependent expansion stage.

Characterization of CD56-/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals.

Natural killer (NK) cells are an important component of the innate immune response against viral infections. NK cell-mediated cytolytic activity is defective in HIV-infected individuals with high levels of viral replication. In the present study, we examined the phenotypic and functional characteristics of an unusual CD56(-)/CD16(+) (CD56(-)) NK subset that is greatly expanded in HIV-viremic individuals. The higher level of expression of inhibitory NK receptors and the lower level of expression of natural cytotoxicity receptors observed in the CD56(-) NK fraction compared with that of CD56(+) NK cells was associated with extremely poor in vitro cytotoxic function of this subset. In addition, the secretion of certain cytokines known to be important in initiating antiviral immune responses was markedly reduced in the CD56(-), as compared with the CD56(+) NK cell subset. These data suggest that the expansion of this highly dysfunctional CD56(-) NK cell subset in HIV-viremic individuals largely accounts for the impaired function of the total NK cell population.

Distress and expression of natural killer receptors on lymphocytes

Chronic distress has been associated with alterations in natural killer (NK) cell and T cell percentages and function. NK cells express inhibitory and stimulatory receptors that regulate cytotoxicity and cytokine secretion. T cells and T cells expressing CD56 (NKT cells) also express these NK-associated receptors, which are thought to serve the same function. The objective of this study was to examine the relationship between distress and expression of NK-associated receptors on NK cells, T cells, and NKT cells. Using multicolor flow cytometry and validated questionnaires, we studied twenty-nine healthy adults with a bimodal age spread. Whereas distress was related to significantly lower percentages of CD3 + T cells, it was related to significantly higher percentages of NKT cells. Distress was associated with significantly higher percentages of T cells expressing NK-associated receptors including CD94 and KIR. In contrast, distress was associated with significantly lower percentages of NK cells bearing KIR (GL183) receptors. Our findings suggest a possible role for NK-associated receptors in distress-related alterations in lymphocyte maturation, trafficking, or activity.

Natural killer T cells and rheumatoid arthritis: friend or foe?

Natural killer T cells and rheumatoid arthritis: friend or foe?

Isolation of CD56+ Natural Killer Cells Using the CliniMACS® MARRS Tubing Set, a Newly Developed Tubing Set Including a Cross-Flow Filtration Module for Removal of Unbound Reagent.

Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers of NK cells in a closed system consisting of clinical grade components. We have previously developed a two step NK cell isolation process in which the CliniMACS® plus Instrument and CliniMACS® CD3 and CD56 Reagents are used for CD3+ cell depletion and subsequent CD56+ cell enrichment, respectively. We have recently optimized the CD3 depletion step for very large scale cell processing by development of the CliniMACS® Depletion Tubing Set (“DTS”) and appropriate cell separation software, reducing the processing time of the CD3 depletion step by 50%. We now have developed the CliniMACS® MARRS Tubing Set (MARRS = MAgnetic Reagent Removal System), a tubing set which includes a cross-flow filtration module for removal of unbound magnetic reagent after magnetic labelling. The cross-flow filtration module allows to directly connect a magnetically labelled cell product to the tubing set without centrifugational washing steps, reducing the processing time by 45 minutes. We isolated CD56+ cells from buffy coats as well as apheresis harvests using the CliniMACS® MARRS Tubing Set as compared to the standard CliniMACS® Tubing Set. Both tubing sets (MARRS vs. standard) yielded CD56+ cells of comparable purity (94.1% vs. 92.3%), comparable depletion of CD56−CD3+ T cells (3.2 log vs. 3.0 log) and comparable recovery (72.9% vs. 76.7%) with a trend towards better performance of the CliniMACS® MARRS Tubing Set. To assess the functionality of CliniMACS® separated CD56+ cells we performed flow cytometric cytotoxicity tests against the MHC class I negative target cell line K562. Specific killing could be increased from <2% (unseparated sample) to 47% (MARRS) and 53% (standard) at an effector to target ratio of 50:1. Thus, K562 killing cells are efficiently enriched and cytotoxicity is similar for both CD56+ cells isolated using the standard CliniMACS® Tubing Set and the CliniMACS® MARRS Tubing Set. We additionally assessed whether the killing efficiency of CD56+ cells correlates with NK receptor expression (NCR, KIR, NKG2D, costimulatory molecules) on different NK and NKT subsets. Results of these analyses will be presented. We conclude that the CliniMACS® MARRS Tubing Set can be used to efficiently isolate functional CD56+ cells from buffy coats or medium scale apheresis harvests (e.g. non-mobilized donors), clearly reducing total processing time. For larger scale NK cell selection using apheresis harvests (e.g. mobilized donors) the combination of Depletion Tubing Set for CD3+ cell depletion and the standard CliniMACS® Tubing Set for CD56+ cell enrichment is suitable.

Prolonged Ex Vivo Expansion (7–10 Days) of Cord Blood (CB) Recryopreserved and Rethawed Aliquots with Anti-CD3, IL-2, IL-12 Significantly Expands NKT Cells, NKT KIR Substes and NK Cytotoxicity Compared to Short-Term Expansion: Potential for CB Adoptive Cellular Immunotherapy (ACI).

CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al, Blood 2002). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood 2001; Locatelli, et al Blood 1999). We have demonstrated the ability to ex-vivo expand (EVE) CB in short term culture (48hrs) with IL-2, IL-7, IL-12 and anti-CD3 (AB/CY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with a significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2/DL2, CD94/NKG2A (Ayello/Cairo et al, BBMT 25a 2004). Timeus et al (Hematologica 2003) demonstrated the ability to sequentially freeze-thaw UCB twice without a detrimental effect on UCB clonogenic potential or viability (Hematologica 2003). In this study we compared previous short term (48hr) cultures with prolonged cultures (48–240hrs) on expansion, maturation, NK and LAK cytolytic ability, KIR inhibitory and C-lectin NK subsets in CTCTE UCB utilizing the same AB/CY cocktail. UCB were cryopreserevd and thawed by the NHBLI/COBLT method (Fraser/Cairo et al, J Hemat 1998). After rethawing, nonadherent UCB cells were cultured in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY] for 4 time periods: 48, 96, 168 and 240 hrs. NK subsets (CD94+, CD16+, CD56+) and NK receptor (KIR3DL1, KIR2DL1/S1, KIR2DL2 and NKG2a) expression were analyzed by flow cytometry using CD3, CD16, CD56, NKBL1, CD158a, CD158b, CD94 and NKG2a mAbs. Additionally, NK and LAK cytotoxicity was measured by europium release assay. There was a significant increase in NKT (CD3+/16+/56+) UCB with AB/CY from 48–240 hrs vs media (2.97±.3 vs 66.7±7.1 vs 35.8±5.9%, p<.001; p<.01), NKT KIR2DL1/S1 from 48–168 hrs vs media (2.76±.4 vs 33.6±8.5 vs 12.1±0.9%, p<.001, p<.05), NKT KIR3DL1 at 48 vs 240 hrs (1.27±0.5 vs 22.2±4.6%, p<.05) and NK (CD3−/16+/56+) KIR3DL1 48 vs 240 hrs vs media (18.8±2.8 vs 38.3±6.3 vs 12.1±4.2%, p<.05, p<.001). A significant increase was shown in UCB NK cytotoxicity on Day 7 (168hrs) [48 vs 96 vs168 hrs] AB/CY vs media alone (168hrs): 53.8±3.9 vs 56±1.6 vs 71.5±.8 vs 40.7±2.0%, p<.001). Similarly, there was a significant maximal increase in UCB LAK cytotoxicity at 168 hrs [48 vs 96 vs 168 hrs] AB/CY vs media: 31.8±1.8 vs 5.47±1.4 vs 63.95±.74 vs 41.6±1.1%, p<.001. We observed a significant enhancement in proliferation at 48 vs 96 vs 168 vs 240 hrs in ABCY vs media (p<.001) and maximal proliferation in ABCY vs media at 240 hrs:.41±.098 vs 3.93±.014, p<.001. This data suggests that UCB cryopreserved cells may be thawed at the time of UCBT, recryopreserved, rethawed at a later date, EVE and activated to yield viable NK and NKT KIR subsets with enhanced cytolytic ability for ACI to induce GVHM effects after UCBT.

Pattern of Ly49A Receptor Downregulation in Low-Level Prenatal Allogeneic Chimeras Suggests Threshold of Chimerism Needed for Long-Term Engraftment.

Despite clinical success with in utero hematopoietic stem cell transplantation in immunodeficient recipients, only microchimerism (<0.1%) has been observed in immunocompetent human or non-human primate hosts without conditioning. It has been postulated that an innate immune response to the low levels of chimerism caused the lack of engraftment. We have found that prenatal murine recipients of fetal allografts with low levels of engraftment (<2.0%) also lose their chimerism after a few months. To better understand fetal innate immunity as it relates to this problem, we studied the education of host NK cells in prenatal chimeras with low engraftment (<5.0%). We hypothesized that alloreactive NK receptor expression correlates with chimerism level. To test this hypothesis, prenatal chimeras were created by transplanting fetal liver light density cells from 14 dpc Balb/c (H2d) fetuses into 14 dpc B6 (H2b) fetuses by IP injection. Using flow cytometry, peripheral blood chimerism in the pups was first assessed at 3 weeks and followed for one year. At 12 weeks, following the acquisition of mature NK inhibitory receptors, the level of H-2d alloreactive Ly49A, C, F, G, and I receptor expression among host splenic NK cells was measured. The mean fluorescence intensity for each receptor relative to age-matched controls (rMFI) was plotted against chimerism and the Pearson correlation coefficient (r) calculated for comparison. Chimerism ranged from 0.1 – 5.0% without GVHD (n=13). As shown in figure 1, B6 recipient NK cells exhibited a dramatic negative correlation of Ly49A expression with increasing chimerism (rMFI = 38 –118%, r = −0.87) without change in the frequency of Ly49A+ NK cells (18–26%, r=0.37). No significant change in the expression of Ly49C, F, G, or I was seen within the range of chimerism analyzed. Five mice reached 12 months and were sacrificed for a repeat NK analysis to determine the stability of Ly49A expression, see table 1. In four mice, chimerism and Ly49A expression levels did not change significantly. However, chimerism was lost in one animal with a corresponding upregulation in Ly49A expression. The initial chimerism level was in a range where we find that very few mice maintain long-term chimerism in this system (<2.0%). The pattern of progressive Ly49A downregulation with increasing peripheral blood chimerism strongly supports a mechanism of NK education that is developmentally responsive to the composite MHC expression in the host chimera. This suggests that a threshold level of chimerism may be necessary to overcome the host innate immune response. Possible mechanisms by which this may occur include higher levels chimerism leading to further NK inhibition or possibly the expansion of a critical regulatory cell population in the donor cell fraction. An understanding of these potential mechanisms will facilitate the development of more effective transplantation regimens.Table 1. Expression of Ly49A at 3 months and 1 yearChimerism LevelLy49A expressionAnimalPB-3 months (%)PB-12 months (%)rMFI-3 months (%)rMFI-12 months (%)78310.20.176747759*1.330.2389278303.221.81452978601.912.38423378384.95.23832 [Display omitted]