High Natural Killer Research Articles

Abstract CT147: Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial

Abstract Background; Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells, and produce numerous cytokines resulting in the activation of the adoptive immune systems. Thus, NK cell is considered ideal cell for adoptive cancer immunotherapy. But the difficulty of obtaining large numbers of NK cells that are safe to administer deters its clinical use. We successfully generated large numbers of activated NK cells from small quantities of blood and also determined that the expanded cells were safe to administer in a monotherapy (J Transl Med (2015) 13:277). Since NK cells play a key role in antibody-dependent cellular cytotoxicity (ADCC), administration of NK cells would be expected to enhance the efficacy of IgG1 monoclonal antibodies by augmenting ADCC. This phase I clinical trial evaluated the safety, toxicity, and immunological response of the adoptive NK therapy in combination with IgG1 antibodies. Patients and Methods; Patients with unresectable advanced gastric or colorectal cancer who have administered or have planned to administer IgG1 antibody (i.e. trastuzumab for HER2 positive gastric cancer or cetuximab for RAS-wild colorectal cancer). NK cells were expanded from PBMCs with the same method in previous clinical trial, briefly ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296-induced T cells. Expanded NK cells were intravenously injected 3 days after IgG1 antibody administration in a dose-escalating manner (dose 0.5 × 109, 1.0 × 109, 2.0 × 109 cells/injection, three patients/one cohort), every 3 weeks for 3 cycles. We evaluated the safety and efficacy of the combination therapy. To assess the immunological response, immunomonitoring was performed. Results; Nine eligible patients were enrolled. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D(97.6%) and CD16(69.6%). This combination therapy was well tolerated with no severe adverse events. Among six evaluable patients, 4 presented SD, 2 presented PD. Of the four SD patients, 3 showed an overall decrease in tumor size after combination therapy. In immunomonitoring analysis, whole blood IFN-gamma production level was increased and the proportion of Treg (Treg/CD4) in peripheral blood was decreased after the combination therapy. Conclusions; This is the first clinical trial of the combination of adoptive NK cell therapy and IgG1 monoclonal antibodies. Our data provide evidence of good tolerability and preliminary anti-tumor activity for this combination therapy. Moreover, immunological monitoring revealed that the combination therapy induced Th1-type immune response and reduced peripheral Tregs. However, given the limited activity observed in this clinical trial, additional studies are necessary to develop these combination approaches. Citation Format: Takeshi Ishikawa, Tetsuya Okayama, Naoyuki Sakamoto, Mitsuko Ideno, Kaname Oka, Tatsuji Enoki, Junichi Mineno, Hideyuki Konishi, Satoshi Kokura, Yuji Naito, Yoshito Itoh. Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT147.

PO-414 Stage IV melanoma patients with tumoural MHC class i loss only respond to anti-PD-1 therapy in the presence of high NK cell density

IntroductionPrior to 2011 there were no effective systemic therapies for advanced stage melanoma patients and the overall survival was 7.5 months. Recently, immune checkpoint inhibitors, anti-PD-1 and anti-CTLA-4 inhibitors, have produced response rates as high as 50% and doubled overall survival, but only 30% of patients have a durable response. One important resistance mechanism to immunotherapies is the downregulation of MHC Class I, resulting in the expansion of melanoma cells resistant to CD8 +T cell killing. Natural Killer (NK) cells monitor MHC Class I expression and eliminate cells that fail to express it; thus, NK cells are likely critical in preventing resistance to anti-PD-1 therapy. This study sought to investigate whether tumour-infiltrating NK cells in the presence of MHC class I loss improved survival outcome of patients treated with anti-PD-1.Material and methodsTwenty-five stage IV metastatic melanoma patients treated with anti-PD-1 therapy were categorised into responders (CR/PR/SD >6 mo, n=13) and non-responders (SD <6 mo/PD, n=12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on formalin-fixed-paraffin embedded pre-treatment tumour samples. Flow cytometry was used to confirm novel NK phenotypes in melanoma lymph node metastases of treatment naïve stage IV melanoma patients (n=5).Results and discussionsDifferential expression analysis identified nine up-regulated NK cell specific genes in responders when compared to non-responders (adjusted p<0.05). Immunofluorescent staining of biopsies confirmed a significantly higher density of intratumoural and peritumoural CD16+ (intratumoural p=0.0015 and peritumoural p=0.0039) and granzyme B+ (intratumoural p=0.019 and peritumoural p=0.011) NK cells in responding patients. Flow cytometry demonstrated 46%±8% of the NK cells were identified as expressing PD-1. When stage IV melanoma patients were further stratified by MHC class I loss, responding patients with MHC class I loss, had a higher NK cell density and better survival outcome, compared to non-responders (p=0.012).ConclusionThis study showed that the presence of higher numbers of NK cells are associated with improved responses to anti-PD-1 therapy. Most importantly, responding patients with MHC class I loss had higher NK cell densities, suggesting that NK cells play an important role in mediating response to anti-PD-1 in patients whose tumour down regulates MHC class I expression.

NK cell and Th17 responses are differentially induced in murine cytomegalovirus infected renal allografts and vary according to recipient virus dose and strain.

Human cytomegalovirus (HCMV) donor positive (D+) serostatus with acute rejection is associated with renal allograft loss, but the impact of recipient positive (R+) serostatus is unclear. In an allogeneic renal transplant model, antiviral natural killer (NK) and CD8+ T cell memory responses in murine CMV (MCMV) D+/R+ transplants were compared to D-/R- and D+/R- transplants, with recipient infection varied by MCMV dose and strain. D+/R- transplants had high primary antiviral cytolytic (interferon-γ+) and cytotoxic (granzyme B+) NK responses, whereas NK memory responses were lower in D+/R+ recipients receiving a high primary MCMV dose. Despite MCMV immunity, D+/R+ recipients receiving a low MCMV dose showed primary-like high cytolytic and cytotoxic NK responses. D+/R+ transplants infected with different D/R strains had low cytolytic NK responses but high cytotoxic NK responses. NK memory also induced a novel TNF-α+ NK response among high-dose virus recipients. MCMV+ transplants had greater Th17 responses than MCMV-uninfected transplants and Th17 inhibition ameliorated graft injury. All MCMV+ recipients had similar CD8+ T cell responses. In sum, NK and Th17 responses, but not CD8+ T cells, varied according to conditions of primary recipient infection. This variability could contribute to variable graft outcomes in HCMV D+/R+ renal transplantation.

Impaired natural killer cell subset phenotypes in human obesity

Obesity is associated with alterations in functionality of immune cells, like macrophages and natural killer (NK) cells, leading to an increased risk for severe infections and several cancer types. This study aimed to examine immune cell populations and functional NK cell parameters focusing on NK cell subset phenotypes in normal-weight and obese humans. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from normal-weight and obese individuals and analyzed by flow cytometry. Results show no significant changes in the frequency of monocytes, B lymphocytes, or NKT cells but a significantly increased frequency of T lymphocytes in obesity. The frequency of total NK cells was unaltered, whereas the number of low cytotoxic CD56bright NK cell subset was increased, and the number of high cytotoxic CD56dim NK cell subset was decreased in obese subjects. In addition, the frequency of CD56bright NK cells expressing the activating NK cell receptor NKG2D as well as intracellular interferon (IFN)-γ was elevated in the obese study group. In contrast, the frequency of NKG2D- and IFN-γ-positive CD56dim NK cells was lower in obesity compared to normal-weight individuals. Moreover, the expression of the activation marker CD69 was decreased in NK cells, which can be attributed to a reduction of CD69-positive CD56dim NK cells in obese subjects. In conclusion, data reveal an impaired NK cell phenotype and NK cell subset alterations in obese individuals. This NK cell dysfunction might be one link to the higher cancer risk and the elevated susceptibility for viral infections in obesity.

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment

IntroductionWe previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second- or subsequent-line dasatinib treatment safely at a median follow-up of 20 months. However, the results from longer follow-up periods would be much more useful from a clinical perspective. Patients and MethodsThe DADI trial was a prospective, multicenter trial conducted in Japan. After confirming a stable DMR for ≥ 1 year, dasatinib treatment subsequent to imatinib or nilotinib was discontinued. After discontinuation, the loss of DMR (even of 1 point) was defined as stringent molecular relapse, thereby triggering therapy resumption. The predictive factors of treatment-free remission (TFR) were analyzed. ResultsThe median follow-up period was 44.0 months (interquartile range, 40.5-48.0 months). The estimated overall TFR rate at 36 months was 44.4% (95% confidence interval, 32.0%-56.2%). Only 2 patients developed a molecular relapse after the 1-year cutoff point. The presence of imatinib resistance was a significant risk factor for molecular relapse. Moreover, high natural killer cell and low γδ+ T-cell and CD4+ regulatory T-cell (CD25+CD127low) counts before discontinuation correlated significantly with successful therapy discontinuation. ConclusionThese findings suggest that discontinuation of second- or subsequent-line dasatinib after a sustained DMR of ≥ 1 year is feasible, especially for patients with no history of imatinib resistance. In addition, the natural killer cell count was associated with the TFR.

Glypican-1 as a novel immunotherapeutic target in prostate cancer.

174 Background: New effective therapies for men with prostate cancer are desperately needed. Recently, cancer immunotherapy has emerged as an important new treatment strategy for prostate cancer and for castrate resistant prostate cancer (CRPC). Multiple studies have identified the heparan sulfate proteoglycan-1 Glypican 1 (GPC-1) as being overexpressed in different cancers, and also as being a possible marker of poor prognosis in several solid tumor cancers. GPC-1 has been recently identified as a potential marker for prostate cancer. The MIL-38 monoclonal antibody detects GPC-1 and an IgG1 chimeric version of this antibody has been developed for preclinical studies. Here we sought to examine MIL-38 binding to a panel of prostate cancer cell lines and examine its feasibility as a novel immunotherapeutic agent targeting GPC-1 in prostate cancer Methods: Expression of GPC-1 in CRPC cell lines was examined by Flow cytometry and Western Blotting using MIL-38 as the detector antibody. The competency of GPC-1 as an immunotherapeutic target was assessed via chimeric MIL-38 induced Antibody Dependent Cell Cytotoxicity (ADCC) using high affinity Natural Killer cells (haNKs) in vitro . Results: Flow cytometry and Western blot assessments of normal prostatic epithelial cells (i.e. RWPE-1) and cells from prostate cancer cell lines (i.e. PC-3, 22RV1, DU-145, VCaP, LNCaP, CWR-R1, and LAPC-4) revealed that only cancer cells expressed GPC-1. Enzalutamide resistant cell lines demonstrated higher expression of GPC-1 than their respective parental line. ADCC assays demonstrated enhanced haNK – prostate cancer cell cytotoxicity in the presence of chimeric MIL-38 anti-GPC-1 antibody, while the IgG1 isotype control had no effect. Conclusions: GPC-1 protein was expressed by most prostate cancer cell lines, including enhanced expression by enzalutamide resistant cells. Preliminary in vitro ADCC assay results revealed the potential utility of GPC-1 as an immunotherapeutic target in prostate cancer.

Immune modulation of i.v. immunoglobulin in women with reproductive failure.

BackgroundThe mechanism of maternal immune tolerance of the semi‐allogenic fetus has been explored extensively. The immune reaction to defend from invasion by pathogenic microorganisms should be maintained during pregnancy. An imbalance between the immune tolerance to the fetus and immune activation to the pathogenic organisms is associated with poor pregnancy outcomes. This emphasizes that the immune mechanism of successful reproduction is not just immune suppression, but adequate immune modulation.MethodsIn this review, the action of i.v. immunoglobulin G (IVIg) on the immune system and its efficacy in reproductive failure (RF) was summarized. Also suggested is the indication of IVIg therapy for women with RF.Main findings (Results)Based on the mechanism of the immune regulation of IVIg and following confirmation of the immune modulation effects of it in various aberrant immune parameters in patients with RF, it is obvious that IVIg is effective in recurrent pregnancy losses and repeated implantation failures with immunologic disturbances.ConclusionThe authors recommend IVIg therapy in patients with RF with aberrant cellular immunologic parameters, including a high natural killer cell proportion and its cytotoxicity or elevated T helper 1 to T helper 2 ratio, based on each clinic's cut‐off values. Further clinical studies about the safety of IVIg in the fetus and its efficacy in other immunologic abnormalities of RF are needed.

SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7+ normal lymphocytes

AbstractSLAMF7 is under intense investigation as a target for immunotherapy in multiple myeloma. In this study, we redirected the specificity of T cells to SLAMF7 through expression of a chimeric antigen receptor (CAR) derived from the huLuc63 antibody (elotuzumab) and demonstrate that SLAMF7-CAR T cells prepared from patients and healthy donors confer potent antimyeloma reactivity. We confirmed uniform, high-level expression of SLAMF7 on malignant plasma cells in previously untreated and in relapsed/refractory (R/R) myeloma patients who had received previous treatment with proteasome inhibitors and immunomodulatory drugs. Consequently, SLAMF7-CAR T cells conferred rapid cytolysis of previously untreated and R/R primary myeloma cells in vitro. In addition, a single administration of SLAMF7-CAR T cells led to resolution of medullary and extramedullary myeloma manifestations in a murine xenograft model in vivo. SLAMF7 is expressed on a fraction of normal lymphocytes, including subsets of natural killer (NK) cells, T cells, and B cells. After modification with the SLAMF7-CAR, both CD8+ and CD4+ T cells rapidly acquired and maintained a SLAMF7– phenotype and could be readily expanded to therapeutically relevant cell doses. We analyzed the recognition of normal lymphocytes by SLAMF7-CAR T cells and show that they induce selective fratricide of SLAMF7+/high NK cells, CD4+ and CD8+ T cells, and B cells. Importantly, however, the fratricide conferred by SLAMF7-CAR T cells spares the SLAMF7–/low fraction in each cell subset and preserves functional lymphocytes, including virus-specific T cells. In aggregate, our data illustrate the potential use of SLAMF7-CAR T-cell therapy as an effective treatment against multiple myeloma and provide novel insights into the consequences of targeting SLAMF7 for the normal lymphocyte compartment.

Immune Abnormalities in Patients With Single Ventricle Circulation Precede the Fontan Procedure.

Immune abnormalities are common in Fontan patients with protein-losing enteropathy. Limited data exist on immune function of other patients with single ventricle circulation. This prospective cohort study evaluated immunologic characteristics of children with single ventricle circulation from neonatal age up to early post-Fontan period. Low leukocyte counts were observed in half of the patients prior to bidirectional Glenn and Fontan surgery. Total lymphocyte counts were below normal range in 36% to 63% of patients across all groups except patients following Fontan procedure who had normal counts. Typical lymphocyte subpopulation patterns were (1) high counts of total and helper T lymphocytes (CD3+ and CD4+ cells), low B lymphocytes (CD19+ cells), and increased CD4/CD8 ratio in neonates and (2) low T lymphocytes (CD3+, CD4+, CD8+ cells) with high natural killer cells (CD16+) and B lymphocytes (CD19+ cells) in other groups. Low preoperative total lymphocyte counts were associated with longer intensive care unit stay in patients after bidirectional Glenn and Fontan procedure ( P = .03 and P = .01, respectively) and low leukocyte counts with higher incidence of pleural effusions and chylothorax after Fontan procedure ( P = .005 and P = .002, respectively). Single ventricle patients display several immunological abnormalities. Beyond the neonatal age, an immune pattern includes CD3+, CD4+, CD8+ lymphopenia, and CD16+ and CD19+ lymphocytosis. B-cell lymphocytosis compensates T-cell lymphopenia, producing normal total lymphocyte counts in patients early after Fontan surgery. Low preoperative total lymphocyte counts may be associated with longer postoperative intensive care unit stay in patients with bidirectional Glenn and Fontan procedure and leukopenia with pleural effusions in Fontan patients.

Association Between Natural Killer Cell Activity and Colorectal Cancer in High-Risk Subjects Undergoing Colonoscopy

Low activity of natural killer (NK) cells has been associated with increased risk of cancer and has been reported in patients with colorectal cancer (CRC). Activity of NK cells can be measured in a small volume of whole blood by a commercially available test. We investigated whether this test could be used to identify patients with CRC, using findings from colonoscopy as a reference standard. We performed an open-label, prospective, cross-sectional study of 872 high-risk subjects (more than 40 years old) screened for CRC by colonoscopy at a university hospital in Montreal, Canada from October 2014 through January 2016. Blood samples were collected on the day of colonoscopy, prior to the procedure. The test involves stimulation of whole blood with cytokine that induces NK cells to secrete interferon gamma (IFNG), which is quantified by an ELISA. Tissue samples were taken from lesions during the colonoscopy and analyzed histologically; subjects were classified as having no evidence of disease, adenomatous polyps of less than 10 mm, of 10 mm or more, or CRC. We used the non-parametric Mann-Whitney test to compare NK cell activity between subjects with no evidence of CRC and subjects found to have CRC. Receiver operating characteristic curve analysis was used to assess the ability of the test to identify individuals with CRC. The primary objective was to determine the difference in NK cell activity between subjects with vs without CRC. The secondary objective was the test performance, based on receiver operating characteristic analysis, and cut-off value that most accurately identified individuals with CRC. We found a significant difference in NK cell activity between the 23 subjects with CRC (based on pathology analysis) and the 849 subjects without CRC: subjects found to have CRC by colonoscopy had a median level of 86.0 pg IFNG/mL (inter-quartile range, 43.3-151.0 pg IFNG/mL), whereas subjects without CRC had a median level of 298.1 pg IFNG/mL (inter-quartile range, 100.4-920.2 pg IFNG/mL) (P= .0002). The cut-off value that most accurately identified subjects with CRC was 181 pg/mL. The NK cell activity test identified subjects with CRC with 87.0% sensitivity, 60.8% specificity, a positive predictive value of 5.7%, and a negative predictive value of 99.4%. The odds ratio for detection of CRC in subjects with low NK cell activity vs subjects with higher NK cell activity was 10.3 (95% CI, 3.03-34.9). Using colonoscopy as the reference standard, a test for NK cell activity in whole blood samples identified patients with CRC with 87.0% sensitivity and a negative predictive value of 99.4%. Subjects with low NK cell activity had a 10-fold higher risk of CRC compared with subjects with high NK cell activity. This test might be used in clinical practice to assess patients for risk of CRC. Clinicaltrials.gov number: NCT02291198.

Combined peripheral natural killer (NK) cell and circulating tumor cell (CTC) enumeration to enhance prognostic efficiency in patients (pts) with triple-negative breast cancer (TNBC).

1105 Background: CTCs have emerged as an independent prognostic factor for metastatic breast cancer. However, the prognostic value of baseline CTCs regarding PFS in TNBC is still controversial, especially beyond first-line. We evaluated a novel combined NK cell/CTC detection system for enumeration to better understand the impact of cell count on prognosis in TNBC. Methods: 83 consecutive patients with metastatic TNBC were enrolled and received a new line of therapy (median=2, range: 1~5). Baseline circulating CTCs and NK cells were isolated and enumerated simultaneously prior to starting a new line of therapy using our previously published method targeting EpCAM (Bai et al. J Mater Chem B, 2014) and flow cytometry (antibodies against CD3-, CD45+, CD56+) respectively. The NK cell level was expressed as the proportion of total lymphocytes (%). Patients with normal to high NK cell proportion (≥ 9.15%) and &gt; 2 CTC/2 ml were defined as NK resistant CTC, the rest were defined as non-NK resistant CTC. Results: 33 out of 83 TNBCs had first-line of therapy. Pts with ≤ 2 CTC/2 ml had a significantly longer median PFS than those with &gt; 2 CTC/2 ml ( P = 0.028). The differences in median PFS were not significant ( P=0.110) for the entire group of pts with TNBC receiving different lines of therapy. Pts with non-NK resistant CTCs had a significantly longer median PFS than those with NK resistant CTCs ( P= 0.025), regardless of line of therapy. Conclusions: Including peripheral NK cell level into consideration can improve CTC prognostic efficiency in predicting PFS for TNBCs receiving different line of therapy. Further analysis of the unique biological features of NK-resistant CTCs and associated clinical consequence holds promise in guiding clinical practice. [Table: see text]

Natural killer cells as a predictive biomarker for response to anti-PD-1 therapy in patients with advanced solid tumors.

e21055 Background: Assessment of PD-L1 expression in tumor biopsy has been widely used for predicting anti-PD-1 therapy. Several factors, including tumor heterogeneity, false negative staining and unavailability of biopsy samples limit the application. Natural killer (NK) cells exert cytotoxicity against cancer cells. Therefore, we explored using NK cells percentage in peripheral blood (PB) as a biomarker to identify pts who are more likely to benefit from anti-PD-1 therapy. Methods: Pts treated with anti-PD-1 antibody (JS001) 1 mg/kg, 3mg/kg or 10mg/kg every 2 weeks in a Phase I clinical trial (ClinicalTrials.gov Identifier: NCT02836795) had PB collected at baseline and at time of radiographic evaluation performed every 8 wks. Disease status (shrinking stable disease, growing stable disease, partial response, complete response, or progressive disease) was characterized using RECIST v1.1 and immune-related response criteria. Frequencies of NK cells (CD3-CD16+CD56+) were measured by flow cytometry. Results: Thirty-six patients were enrolled in this trial, including 22 melanoma, 9 urothelial carcinoma and 5 renal cell carcinoma. As of 1/27/2017, median treatment duration: 3.92 months. Radiographic tumor evaluation has been performed in 32 pts: 1/32 CR, 6/32 PR, 6/ 32 shrinking SD, 4/32 growing SD and 15/32 PD. Pts with high NK cells at baseline observed 60% (6/10) Clinical Benefit Rate (CR/PR/ shrinking SD) versus 40% (13/32) in overall pts. Patients with clinical benefit had higher frequency of NK cells at baseline compared to pts with radiographic growing SD and PD (mean 24.3% vs. 17.9%, P = 0.076). Furthermore, pts with NK cell level consistently above UN or increasing above UN during treatment are more likely to get clinical benefit than those under UN (7/10 vs 3/22, P = 0.013). Conclusions: Baseline high NK cells frequency may predict a favorable response to anti-PD-1 therapy, which provides a non-invasive way to monitor response to PD-1 blockade. These results are being validated in a phase II trial. Clinical trial information: NCT02836795.

Association of peripheral NK cell counts with Helios+ IFN-γ- Tregs in patients with good long-term renal allograft function.

Little is known about a possible interaction of natural killer (NK) cells with regulatory T cells (Treg ) in long-term stable kidney transplant recipients. Absolute counts of lymphocyte and Treg subsets were studied in whole blood samples of 136 long-term stable renal transplant recipients and 52 healthy controls using eight-colour fluorescence flow cytometry. Patients were 1946 ± 2201 days (153-10268 days) post-transplant and showed a serum creatinine of 1·7 ± 0·7 mg/dl. Renal transplant recipients investigated >1·5 years post-transplant showed higher total NK cell counts than recipients studied <1·5 years after transplantation (P = 0·006). High NK cells were associated with high glomerular filtration rate (P = 0·002) and low serum creatinine (P = 0·005). Interestingly, high NK cells were associated with high CD4+ CD25+ CD127- forkhead box protein 3 (FoxP3+ ) Treg that co-express the phenotype Helios+ interferon (IFN)-γ- and appear to have stable FoxP3 expression and originate from the thymus. Furthermore, high total NK cells were associated with Treg that co-express the phenotypes interleukin (IL)-10- transforming growth factor (TGF)-β+ (P = 0·013), CD183+ CD62L- (P = 0·003), CD183+ CD62+ (P = 0·001), CD183- CD62L+ (P = 0·002), CD252- CD152+ (P < 0·001), CD28+ human leucocyte antigen D-related (HLA-DR- ) (P = 0·002), CD28+ HLA-DR+ (P < 0·001), CD95+ CD178- (P < 0·001) and CD279- CD152+ (P < 0·001), suggesting that these activated Treg home in peripheral tissues and suppress effector cells via TGF-β and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). The higher numbers of NK and Treg cell counts in patients with long-term good allograft function and the statistical association of these two lymphocyte subsets with each other suggest a direct or indirect (via DC) interaction of these cell subpopulations that contributes to good long-term allograft acceptance. Moreover, we speculate that regulatory NK cells are formed late post-transplant that are able to inhibit graft-reactive effector cells.

Single-Agent MOR208 in Relapsed or Refractory (R-R) Non-Hodgkin's Lymphoma (NHL): Results from Diffuse Large B-Cell Lymphoma (DLBCL) and Indolent NHL Subgroups of a Phase IIa Study

Single-Agent MOR208 in Relapsed or Refractory (R-R) Non-Hodgkin's Lymphoma (NHL): Results from Diffuse Large B-Cell Lymphoma (DLBCL) and Indolent NHL Subgroups of a Phase IIa Study

Early Natural Killer Cell Reconstitution Predicts Overall Survival in T Cell–Replete Allogeneic Hematopoietic Stem Cell Transplantation

Early immune reconstitution plays a critical role in clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Natural killer (NK) cells are the first lymphocytes to recover after transplantation and are considered powerful effector cells in HSCT. We aimed to evaluate the clinical impact of early NK cell recovery in T cell–replete transplant recipients. Immune reconstitution was studied in 298 adult patients undergoing HSCT for acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome from 2005 to 2013. In multivariate analysis NK cell numbers on day 30 (NK30) > 150 cells/µL were independently associated with superior overall survival (hazard ratio, .79; 95% confidence interval, .66 to .95; P = .01). Cumulative incidence analyses showed that patients with NK30 > 150 cells/µL had significantly less transplant-related mortality (TRM), P = .01. Patients with NK30 > 150 cells/µL experienced significantly lower numbers of life-threatening bacterial infections as well as viral infections, including cytomegalovirus. No association was observed in relation to relapse. These results suggest an independent protective effect of high early NK cell reconstitution on TRM that translates into improved overall survival after T cell–replete HSCT.

Is immunotherapy useful in recurrent implantation failure patients; especially with high natural killer cell?

IVIG is an established treatment in several autoimmune and inflammatory diseases and has been tested in several RCTs in recurrent miscarriage (RM) patients. Although clinically different disorders, RIF has been suggested to share many characteristics with early RM. Immunological parameters that seem associated with both RIF and RM are increased numbers of peripheral blood natural killer cells. There were several reviews of IVIG efficacy in RM patients with high NK cell, but there were few studies in RIF patients.

Abstract 3245: IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas

Abstract Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded NK and CD8+ T cells. In examining natural killer (NK) cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 breast tumor-bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors. Citation Format: Peter S. Kim, Anna R. Kwilas, Wenxin Xu, Sarah Alter, Emily K. Jeng, Hing C. Wong, Jeffrey Schlom, James W. Hodge. IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3245.

IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity against murine breast and colon carcinomas.

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded natural killer (NK) and CD8+ T cells. In examining NK cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1−) phenotype. In 4T1 breast tumor–bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells and CD8+ T cell responders of the innate phenotype, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.

T and NK Cell Phenotypic Abnormalities in Systemic Sclerosis: a Cohort Study and a Comprehensive Literature Review.

Scleroderma (SSc) is a rare and heterogeneous immune-mediated disease involving the connective tissue and microvasculature whose pathogenesis remains unclear. Data concerning T and natural killer (NK) cell abnormalities and cytokine levels in the peripheral blood (PB) from patients with SSc are scarce, and the results are contradictory. The present study aimed to analyze the changes of T lymphocytes, NK cells, and T helper (Th)-related cytokines in the PB of patients with SSc in comparison to healthy individuals and its relation to disease subtype and stage, organ involvement, and nailfold capillaroscopic changes. A non-random convenience sample of 57 scleroderma patients was utilized. Fifty-five out of the 57 patients studied were women (97 %); 10 patients presented pre-scleroderma (pre-SSc) and 47 SSc: 34 limited cutaneous SSc (lcSSc) and 13 diffuse cutaneous SSc (dcSSc). Patients with SSc were classified in early (n = 7), intermediate (n = 10), and late (n = 30) disease. Blood samples were analyzed by flow cytometry for total T cells, CD4+ and CD8+ T cell subsets, total NK cells, and CD56+low and CD56+high NK cell subsets. T cells were further analyzed for the expression of the CD56 adhesion molecule and activation-related markers (HLA-DR, CD45RO). In addition, the serum levels of Th1-, Th2-, and Th17-related cytokines were measured by flow cytometry. Twenty-five healthy individuals recruited from the blood bank were used as controls. Patients had lower numbers of total lymphocytes and T cells comparing to healthy controls. Both CD4+ and CD8+ T cells were decreased, but differences were statistically significant only for CD8+ and CD8+ CD45RO+ T cells. These alterations were seen in patients with SSc but not in patients with pre-SSc, and, in general, they were more pronounced in patients with dcSSc than in patients with lcSSc, in patients with vascular involvement than in those without, as well as in patients having active and late nailfold capillaroscopic patterns. CD56+ T cells were also decreased in SSc patients, especially in those with active/late capillaroscopic patterns or with severe lung disease. Diminished numbers of circulating NK cells were also observed in patients with lcSSc and in those with early disease. No statistically significant changes were found in serum cytokine levels, as compared with controls. Patients with SSc had major alterations in circulating CD8+ and CD56+ T cells, as well as in NK cells, suggesting that these cells may play a relevant role in SSc pathogenesis, probably operating at different phases and/or at different organs. In addition, the serum levels of Th1, Th2, and Th17 cytokines did not provide useful information for evaluating T cell polarization in SSc.

Low Counts of B Cells, Natural Killer Cells, Monocytes, Dendritic Cells, Basophils, and Eosinophils are Associated withPostengraftment Infections after Allogeneic Hematopoietic Cell Transplantation.

Hematopoietic cell transplant (HCT) recipients are immunocompromised and thus predisposed to infections. We set out to determine the deficiency of which immune cell subset(s) may predispose to postengraftment infections. We determined day 28, 56, 84, and 180 blood counts of multiple immune cell subsets in 219 allogeneic transplant recipients conditioned with busulfan, fludarabine, and Thymoglobulin. Deficiency of a subset was considered to be associated with infections if the low subset count was significantly associated with subsequent high infection rate per multivariate analysis in both discovery and validation cohorts. Low counts of monocytes (total and inflammatory) and basophils, and low IgA levels were associated with viral infections. Low plasmacytoid dendritic cell (PDC) counts were associated with bacterial infections. Low inflammatory monocyte counts were associated with fungal infections. Low counts of total and naive Bcells, total and CD56(high) natural killer (NK) cells, total and inflammatory monocytes, myeloid dendritic cells (MDCs), PDCs, basophils and eosinophils, and low levels of IgA were associated with any infections (due to any pathogen or presumed). In conclusion, deficiencies of Bcells, NK cells, monocytes, MDCs, PDCs, basophils, eosinophils, and/or IgA plasma cells appear to predispose to postengraftment infections.