Peripheral Natural Killer Cells Research Articles

Peripheral NK cells identified as the predictor of response in extensive-stage small cell lung cancer patients treated with first-line immunotherapy plus chemotherapy.

Although immunotherapy improves outcomes in extensive-stage small-cell lung cancer (ES-SCLC), the search for biomarkers predicting treatment success is crucial. Natural killer (NK) cells are potential indicators in various cancers, however, their precise role in ES-SCLC prognosis remains unclear. In this retrospective study, 33 patients with ES-SCLC treated with first-line immuno-chemotherapy were enrolled. The peripheral NK cell percentage and its longitudinal dynamics were analyzed using flow cytometry. Progression-free survival (PFS) and overall survival (OS) were calculated as hazard ratio (HR) and compared statistically. The median PFS was better in the group with normal baseline NK cell levels than the low group (7.0 vs. 4.6months; HR = 0.17; 95% CI 0.07-0.41; P < 0.0001), but there was no association with OS (14.9 vs. 10.3months; HR = 0.55; 95% CI 0.23-1.31; P = 0.171). Furthermore, the NK cell% for 95.0% of patients increased after immunochemotherapy in the clinical response group (P = 0.0047), which led to a better median PFS (6.3 vs. 2.1months; HR = 0.23; 95% CI 0.05-0.98; P < 0.0001) and OS (14.9 vs. 5.9months; HR = 0.20; 95% CI 0.04-1.02; P < 0.0001). Similar trends were observed with NK cell% changes up to disease progression, improving PFS (6.5 vs. 4.3; HR = 0.41; 95% CI 0.12-0.92; P = 0.0049) and OS (17.4 vs. 9.7; HR = 0.42; 95% CI 0.17-1.02; P < 0.0001). In patients with ES-SCLC, the percentage and changes in peripheral NK cells can predict the response to combined immunotherapy and chemotherapy.

Interferon-Gamma Secretion Is Significantly Decreased in Stage III Breast Cancer Patients.

Even though some studies have shown possible clinical relationship between molecular subtypes and tumor infiltrating natural killer (NK) cells around tumors, there are few studies showing the clinical relevance of peripheral NK cell activity at diagnosis in female patients with invasive breast cancer. A total of 396 female invasive breast cancer patients who received curative surgical treatment from March 2017 to July 2021 were retrospectively analyzed. NK cell activation-induced interferon-gamma (IFN-γ) secretion measured by enzyme-linked immunosorbent assay was used to measure the activity of peripheral NK cells. Statistical analyses were performed to determine clinical relationships with major clinicopathologic parameters. Quadripartite NK cell activity measured by induced interferon-gamma showed significant relevance with staging and body mass index, and some of the inflammatory serological markers, namely N/L (neutrophil/lymphocyte), P/N (platelet/neutrophil), and P/L (platelet/lymphocyte), showed significantly different NK activity in each interval by univariate analysis. A binary subgroup analysis, setting the IFN-γ secretion cut-off at 100 pg/mL, showed that stage III was significantly increased and axillary lymph node metastasis positivity was increased in the group of IFN-γ < 100 pg/mL, and IFN-γ secretion decreased with an increasing N stage, increased BMI (body mass index), and decreased production of IFN-γ. Following this, the same binary analysis, but with the IFN-γ secretion cut-off at 250 pg/mL, also showed that secretion in stage III was increased in those concentrations with <250 pg/mL, axillary lymph node positivity appeared to be correlated, and BMI ≥ 30 increased in prevalence. Additional ANOVA post hoc tests (Bonferroni) were performed on some serological markers that tended to be somewhat inconsistent. By subgroup analysis with Bonferroni adjustment between the IFN-γ secretion and TNM stage, no significant difference in IFN-γ secretion could be identified at stages I, II, and IV, but at stage III, the IFN-γ secretion < 100 pg/mL was significantly higher than 250 ≤ IFN-γ secretion < 500 pg/mL or IFN-γ secretion ≥ 500 pg/mL. According to this study, stage III was significantly associated with the lowest IFN-γ secretion. Compared to a higher level of IFN-γ secretion, a lower level of IFN-γ secretion seemed to be associated with increased body mass index. Unlike when IFN-γ secretion was analyzed in quartiles, as the IFN-γ secretion fell below 100 pg/mL, the correlation between axillary lymph node positivity and increased N stage, increased BMI, and increased N/L and P/L, which are suggested poor prognostic factors, became more pronounced. We think a peripheral IFN-γ secretion test might be convenient and useful tool for pretreatment risk assessment and selecting probable candidates for further treatment such as immunotherapy in some curable but high-risk invasive breast cancer patients, compared to other costly assaying of tissue NK cell activity at diagnosis.

Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5β-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

The Ratio of Peripheral Blood Natural Killer Cells is not a Solid Surrogate Immune Index in Unexplained Recurrent Miscarriage.

Immunotherapies targeting peripheral natural killer (pbNK) cells in unexplained recurrent miscarriage (uRM) remain controversial. We hypothesized that the change in pbNK cell count might be a result of innate immune responses rather than a cause. To explore whether the pbNK count is significantly different in women testing positive than those testing negative for commonly studied autoimmune markers. Peripheral blood samples were collected from 302 eligible patients with uRM for the antinuclear antibody (ANA) testing determined by the enzyme-linked immunosorbent assay (ELISA), anti-thyroid peroxidase antibody (TPO-Ab) testing and anti-thyroglobulin antibody (Tg-Ab) testing determined by the chemiluminescent immunoassay, and pbNK cell testing determined by flow cytometry. The patients were divided into two groups according to the pbNK normal range, and the comparative analysis entailed an examination of the prevalence rates of autoantibodies within the high pbNK group and the normal pbNK group, followed by a comprehensive investigation into the potential correlations between autoantibodies and pbNK cells. There was a positive association between TPO-Ab positivity and high pbNK cells (p=0.016, OR=5.097, 95% CI 1.356-19.159), while there was a negative association between ANA positivity and high pbNK cells (p=0.013, OR=0.293, 95% CI 0.111-0.773). TPO-Ab-positive patients had a higher pbNK cell count compared with TPO-Ab-negative patients, while ANA-positive patients had a lower pbNK cell count compared with ANA-negative patients. The change in pbNK cell count may be a consequence of immune responses, and there should be careful consideration in applying it as an immunotherapeutic index.

Ozanimod-mediated remission in experimental autoimmune encephalomyelitis is associated with enhanced activity of CNS CD27low/- NK cell subset.

Ozanimod (RPC1063) is an immunomodulator that has been recently approved by the FDA (2020) for the treatment of relapsing-remitting multiple sclerosis (RRMS). It is a selective agonist of the sphingosine-1-phophate receptors 1 and 5, expressed on naïve and central memory T and B cells, as well as natural killer (NK) cells, and is involved in lymphocyte trafficking. Oral administration of ozanimod was reported to result in rapid and reversible reduction in circulating lymphocytes in multiple sclerosis (MS) patients, however, only minimal effect on NK cells was observed. In this study, we sought to investigate the effect of ozanimod on NK cells and assess whether they play any role in ozanimod-induced remission in experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Active EAE induction was done in C57BL/6 female mice, followed by daily oral treatment with ozanimod (0.6mg/kg) starting at disease onset (score 1). Flow cytometry of blood and CNS was performed 24 hours after the last oral dose of ozanimod treatment in diseased mice. Histological analysis of lumbar spinal cord was performed for evaluating the level of inflammation and demyelination. Depletion of peripheral NK cells was done using anti-NK1.1 mouse antibody (mAb) at day 5 post-EAE induction. Ozanimod was effective in reducing the clinical severity of EAE and reducing the percentage of autoreactive CD4+ and CD8+ T cells along with significant inhibition of lymphocyte infiltration into the spinal cord, accompanied by reversed demyelination. Furthermore, ozanimod treatment resulted in a significant increase in the frequency of total NK cells in the blood and CNS along with upregulation of the activating receptor NKG2D on CD27low/- NK cell subset in the CNS. The effectiveness of ozanimod treatment in inhibiting the progression of the disease was reduced when NK cells were depleted using anti-NK1.1 mAb. The current study demonstrated that ozanimod treatment significantly improved clinical symptoms in EAE mice. Ozanimod and anti-NK1.1 mAb appear to function in opposition to one another. Collectively, our data suggest that ozanimod-mediated remission is associated with an increased percentage of total NK cells and CD27low/- NK cells expressing the activating receptor, NKG2D in the CNS.

The Impact of Cytomegalovirus Infection on Natural Killer and CD8+ T Cell Phenotype in Multiple Sclerosis.

Multiple sclerosis (MS) is a debilitating neurological disease that has been classified as an immune-mediated attack on myelin, the protective sheath of nerves. Some aspects of its pathogenesis are still unclear; nevertheless, it is generally established that viral infections influence the course of the disease. Cytomegalovirus (CMV) is a major pathogen involved in alterations of the immune system, including the expansion of highly differentiated cytotoxic CD8+ T cells and the accumulation of adaptive natural killer (NK) cells expressing high levels of the NKG2C receptor. In this study, we evaluated the impact of latent CMV infection on MS patients through the characterization of peripheral NK cells, CD8+ T cells, and NKT-like cells using flow cytometry. We evaluated the associations between immune cell profiles and clinical features such as MS duration and MS progression, evaluated using the Expanded Disability Status Scale (EDSS). We showed that NK cells, CD8+ T cells, and NKT-like cells had an altered phenotype in CMV-infected MS patients and displayed high levels of the NKG2C receptor. Moreover, in MS patients, increased NKG2C expression levels were found to be associated with higher EDSS scores. Overall, these results support the hypothesis that CMV infection imprints the immune system by modifying the phenotype and receptor repertoire of NK and CD8+ T cells, suggesting a detrimental role of CMV on MS progression.

Human Natural Killer Cells Cryopreserved without DMSO Sustain Robust Effector Responses.

Natural killer (NK) cell-based immunotherapy has benefitted from the multiple strengths that NK cells offer in adoptive transfer settings, not the least of which is their safety and potential for allogeneic use. Such use, however, necessitates the cryopreservation of NK cell-based therapy products to support logistical efforts in deploying these cells in different locations, decentralized from the point of collection or manufacturing. DMSO, the most commonly used cryoprotective agent (CPA), has been effective in protecting immune cells during freezing and thawing, but its ability to induce molecular and genetic changes to immune cells as well as its toxicity has stimulated interest in alternative CPAs. However, replacing DMSO's ability to act intracellularly has been difficult, and the sensitivity of human peripheral blood-derived NK cells to freezing and thawing-induced damage has meant that investigations into the potential of replacing DMSO are lacking. As a first step toward establishing the feasibility of cryopreserving human NK cells with CPAs' alternative to DMSO, we investigate the potential of using noncell-penetrating and cell-penetrating CPAs to recover NK cells post-thaw without DMSO. Here, we find that cryoprotection using cell-penetrating CPAs can retain the viability of human peripheral blood-derived NK cells to a comparable degree to DMSO. In addition, non-DMSO-cryopreserved human NK cells were as cytotoxic as those cryopreserved with DMSO and displayed a comparable level of surface markers of activation. In summary, we present the first example of the potential of developing non-DMSO CPA formulations that could be deployed in future cell therapy regimens.

Distinct peripheral T-cell and NK-cell profiles in HGBL-MYC/BCL2 vs patients with DLBCL NOS

AbstractPatients with high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBL-MYC/BCL2) respond poorly to immunochemotherapy compared with patients with diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) without a MYC rearrangement. This suggests a negative impact of lymphoma-intrinsic MYC on the immune system. To investigate this, we compared circulating T cells and natural killer (NK) cells of patients with HGBL-MYC/BCL2 (n = 66), patients with DLBCL NOS (n = 53), and age-matched healthy donors (HDs; n = 16) by flow cytometry and performed proliferation, cytokine production, and cytotoxicity assays. Compared with HDs, both lymphoma subtypes displayed similar frequencies of CD8+ T cells but decreased CD4+ T cells. Regulatory T-cell (Treg) frequencies were reduced only in patients with DLBCL NOS. Activated (HLA-DR+/CD38+) T cells, PD-1+CD4+ T cells, and PD-1+Tregs were increased in both lymphoma subtypes, but PD-1+CD8+ T cells were increased only in HGBL-MYC/BCL2. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of senescent T cells than HDs. Functional assays showed no overt differences between both lymphoma groups and HDs. Deeper analyses revealed that PD-1+ T cells of patients with HGBL-MYC/BCL2 were exhausted with impaired cytokine production and degranulation. Patients with DLBCL NOS, but not patients with HGBL-MYC/BCL2, exhibited higher frequencies of NK cells expressing inhibiting receptor NKG2A. Both lymphoma subtypes exhibited lower TIM-3+– and DNAM-1+–expressing NK cells. Although NK cells of patients with HGBL-MYC/BCL2 showed less degranulation, they were not defective in cytotoxicity. In conclusion, our results demonstrate an increased exhaustion in circulating T cells of patients with HGBL-MYC/BCL2. Nonetheless, the overall intact peripheral T-cell and NK-cell functions in these patients emphasize the importance of investigating potential immune evasion in the microenvironment of MYC-rearranged lymphomas.

Optimisation of a primary human CAR-NK cell manufacturing pipeline.

Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived Tcells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

Sufficiently activated mature natural killer cells derived from peripheral blood mononuclear cells substantially enhance antitumor activity.

Peripheral blood-derived natural killer (NK) cells spontaneously lyse tumor cells without prior sensitization. However, NK cells in peripheral blood (PBNK cells) are in a resting state and exhibit inhibitory phenotypes and impaired cytotoxicity. Thus, strengthening the cytotoxic effector function of PBNK cells and improving NK cell expansion in vitro for a convenient allogeneic therapy are essential. Pure cytokine activation and expansion of NK cells (super NK [SNK])from peripheral blood mononuclear cells were studied. Markers of activated and inhibited NK cells and cytokine secretion by NK cells were examined using flow cytometry. NK cell antitumor activity in vitro was assessed using lactate dehydrogenase (LDH) cytotoxicity assay and an Incucyte real-time imaging system. Additionally, the function of SNK cells against ascites caused by ovarian cancer in NOD-Prkdc(em26Cd52)il2rg(em26Cd22)/Nju (NCG)mice was determined. In a further investigation of the differences between PBNK and SNK, the mRNA of both cells was sequenced and analyzed. Human peripheral blood mononuclear cells showed selective NK cell expansion upon cytokine activation and culture. Both SNK and PBNK cells expressed activation markers, but at different levels, and SNK cells secreted more cytokines related to cytotoxicity than PBNK cells did. Accordingly, SNK cells exhibited strong antitumor activity ex vivo and improved NCG mice survival after intraperitoneal ovarian cancer transplantation. Mechanistically, SNK cells expressed more genes associated with nucleotide metabolism, fatty acid, and ATP metabolism than PBNK cells. SNK cells derived from peripheral blood mononuclear cells have sufficiently activated mature characteristics and high antitumor activity, rendering them a highly promising and essential therapeutic approach for cancer treatment.

ARL67156, a small-molecule CD39 inhibitor, enhances natural killer cell cytotoxicity against gastric cancer cells in vitro and in nude mice

To investigate the effect of ARL67156, a small-molecule inhibitor of CD39, on cytotoxicity of natural killer (NK) cells against gastric cancer cells. Human peripheral blood-derived primary NK cells isolated and purified using a magnetic bead antibody method were treated with 100 μmol/L ARL67156 for 24 h, and the signaling pathway of NK cell activation was detected by Western blotting. The level of interferon-γ (IFN-γ) in the supernatant of NK cells co-cultured with gastric cancer cells was detected using ELISA, and NK cell CD107a degranulation was measured with flow cytometry. The cytotoxicity of NK cells against co-cultured gastric cancer cells was evaluated using flow cytometry. In a nude mouse model bearing subcutaneous gastric cancer xenografts, the therapeutic effect of intravenous transfusion of NK cells and intraperitoneal injection of ARL67156 was assessed by measuring the changes in tumor volume. (25.97 ± 5.69) % of peripheral blood NK cells from healthy individuals positive for CD39 expression. Treatment with ARL67156 significantly upregulated the activation molecules including NKG2D, DAP10, CD57, and CD16 and reduced the expressions of the inhibitory receptors TIGIT and KIR, thereby promoting the secretion of IFN-γ and CD107a degranulation in NK cells (P < 0.05). In both the in vitro and in vivo experiments, ARL67156 significantly enhanced the cytotoxicity of NK cells against gastric cancer cells (P < 0.05). ARL67156 activates NK cells through the vav1-Syk signaling pathway to enhance their cytotoxicity against gastric cancer cells, which may serve as a new strategy for NK cell immunotherapy for gastric cancer.

Establishment of Cut-off Values for Uterine and Peripheral Blood Natural Killer Cells During the Peri-implantation Period in Fertile Controls and Women with Unexplained Recurrent Implantation Failure.

The purpose of the study was to determine the cut-off values for peripheral and uterine natural killer (pNK, uNK) cells in fertile controls and in women with recurrent implantation failure (RIF). In this study, 50 women with RIF and 50 fertile controls were enrolled. Midluteal endometrial biopsy samples from both cases and controls were obtained for CD 56+ cell immunohistochemistry labeling to identify uNK cells. Peripheral venous blood was also taken during the biopsy to detect pNK cells in peripheral blood mononuclear cells using flow cytometry. Cut-off values were obtained from fertile controls. Using a non-parametric Mann-Whitney U-test, the medians of the data sets were compared. The median values for uNK and pNK cell levels in the control group were 7% and 11.6%, respectively. The median value for uNK cells in RIF patients was 9%, which was higher than the one in controls but not statistically significant (p-value of 0.689). The median pNK levels (11.6% vs. 12.4%) were comparable between the RIF group and the controls. Moreover, it was found that 68% of individuals had uNK cell counts below the reference value, while 32% had excessive levels exceeding 7%. Additionally, only 51.4% of the RIF group had increased pNK cells. The pNK cell cut-off values need to be used with caution because there was no difference between fertile controls and RIF women. If immunotherapy is recommended for RIF women, uNK cell testing should be used as the preferred approach.

Association of recurrent implantation failure and recurrent pregnancy loss with peripheral blood natural killer cells and interferon-gamma level.

Fetal uterine survival depends on maintaining an immune balance between the mother and fetus. This study aimed to investigate the correlation of blood peripheral natural killer (NK) cells and interferon-gamma (IFN-γ) with recurrent recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). In this case-control study, peripheral blood samples were obtained from three groups of RPL, RIF, and parous women without a history of abortion or infertility problems and analyzed by lymphocyte-based flow cytometry. Afterward, the levels of NK cells and IFN-γ were determined. All data were analyzed using one-way analysis of variance and nonparametric Kruskal-Wallis tests. The level of IFN-γ in the RPL group was significantly higher than that in parous women and the RIF group (P<0.05), whereas its level in the RIF group was not significantly different from the control group (P>0.05). A significant correlation was found between the levels of IFN-γ and NK cells in the RPL group (r=0.481; P=0.02). However, no significant correlation was found between the levels of IFN-γ and the active NK cells in the RPL group (P=0.08). Moreover, no significant correlation was found between the levels of NK cells (whether activated or not) and IFN-γ in the RIF patients (P>0.05). Immune dysfunction may not be involved in implantation failure during IVF but may be involved in recurrent miscarriage, probably by increasing IFN-γ levels.

Prognostic impact of absolute peripheral blood NK cell count after four cycles of R-CHOP-like regimen treatment in patients with diffuse large B cell lymphoma

As a subtype of lymphocyte, natural killer (NK) cell is the first line of defense that shows a strong function in tumor immunotherapy response and clinical outcomes. The current study aims to investigate the prognostic influence of peripheral blood absolute NK cell count after four cycles of rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) treatment (NKCC4) in diffuse large B cell lymphoma (DLBCL) patients. A total of 261 DLBCL patients treated with R-CHOP from January 2018 to September 2022 were enrolled. The low NKCC4 was observed in patients who died during the study period compared with survival individuals. A NKCC4 < 135 cells/μl had a remarkable negative influence in overall survival and progression-free survival (PFS) compared to a NKCC4 ≥ 135 cells/μl (p < 0.0001 and p < 0.0004, respectively). In addition, the OS and PFS were synergistically lower in a NKCC4 < 135 cells/μl group among DLBCL patients with GCB type or high IPI. In conclusion, this study indicates NCKK4 as a valuable marker in clinical practice and provides an insight for combination treatment of R-CHOP to improve outcomes of DLBCL patients.

Diagnostic significance of blood lymphocyte activation markers in pre-eclampsia.

The adaptive and innate immune system is important in both initiating and preventing functional disorders during pregnancy, one of which is pre-eclampsia. The research aims to conduct the comparative quantification of selected subpopulations of peripheral blood immunoregulatory cells in pregnant women with pre-eclampsia in the third trimester. The marker receptors CD4, CD8, CD95, CD25, and CD27 and the marker antigen HLA-DR were considered. The screening was performed by flow cytometry with dual phenotyping using phycoerythrin- and fluorescein-isothiocyanate-labeled monoclonal antibodies. Data processing consisted in calculating a likelihood value to assess the statistical significance of the difference between the samples. A statistically significant decrease in the subpopulation titer of T and B lymphocytes with marker receptors CD4, CD8, and CD19 was found in pre-eclampsia patients. In the CD4 carrier T-lymphocyte population, there was an increased expression of the CD25/CD95 activation and apoptosis markers. In the CD8 T-killer population, a decreased representation of the CD27/CD25/CD95 markers of differentiation, activation, and apoptosis was deterministic. The expression pattern of the major histocompatibility complex antigen HLA-DR did not change significantly in normality and pathology. The titer of peripheral natural killer cells carrying the CD56 marker increased in patients with various degrees of disease severity, while the number of CD16 natural killer remained at the level of the control group. The research results suggest that a change in the ratio of the above receptors is a diagnostic indicator for pre-eclampsia.

Decrease in peripheral natural killer cell level during early pregnancy predicts live birth among women with unexplained recurrent pregnancy loss: a prospective cohort study

BACKGROUNDPrevious studies have suggested that the trophoblast cells inhibit the proliferation of peripheral natural killer (pNK) cells and the level of pNK cells decrease in middle and late pregnancy stage among healthy women. The change of pNK cells level during early pregnancy and the relationship between the change of pNK level and pregnancy outcomes in women with unexplained recurrent pregnancy loss (URPL) has not been sufficiently explored. OBJECTIVEThis study aims to characterize the level of pre-pregnancy pNK to early pregnancy among women with URPL and whether the change in the level of pNK cells in early pregnancy from pre-pregnancy can predict pregnancy outcomes. STUDY DESIGNIn this prospective cohort study, 1758 women with recurrent pregnancy loss were recruited between January 2017 and December 2021, of which 252 URPL women had pre-pregnancy and early pregnancy (4-6 weeks gestation) pNK measurements. These 252 women were divided into two groups: those with a lower gestational pNK levels (Group 1) compared to pre pregnancy, and those without (Group 2). Their respective outcomes on live birth and pregnancy loss were comparatively analyzed using Chi-square and Student’s t test. Candidate influence factors for live birth were selected using the Akaike information criterion (AIC). Then the participates were randomly divided into training and testing groups. Multivariable logistic regression model was performed, and nomogram was calculated to assess the possibility of live birth. Predictive accuracy was discriminated by area under the receiver operating characteristic curve (ROC), validated by plotting the predicted probabilities and the observed probabilities. Hosmer-Lemeshow test was used to assess the goodsess of fit. RESULTSWhen early gestational pNK cells levels were compared with pre pregnancy pNK cells levels, 61.5% (154) women had a comparatively lower early gestational pNK cells levels versus 38.9% (98) women with increase or no change of their pNK cells levels. The live birth rate in Group 1 was 89.0% (137/154), which was significantly higher than 49.0% (48/98) of Group 2 (p<0.001). Decrease of pNK cells level (odds ratio [OR]=1.36, 95% CI 1.22-1.55, p<0.001) and anti-Muellerian hormone (AMH) (OR=1.41, 95% CI 1.14-1.81, p=0.003) were important predicting factors for higher live birth. Female BMI (OR=0.97, 95% CI 0.82-1.15, p=0.763) and parity (OR=1.61, 95% CI 0.71-4.12, p=0.287) were also predicting factors. Furthermore, the area under the ROC curve of the model to diagnose of live birth was 0.853 with a sensitivity of 81.6% and specificity of 78.0% using the training dataset. And the Hosmer−Lemeshow test showed that the model was a good fit (p = 6.068). CONCLUSIONWe report a comparative decrease in pNK cells levels in over 60% URPL women at 4-6 weeks of gestation, when compared to their pre-pregnancy pNK cells level. Compared to pre pregnancy pNK cells levels, a decrease pNK cells level during early pregnancy might be a useful predictor for LBR in women with URPL.

Acute toxicities of intravenous, intraperitoneal, or intratumoral injection of natural killer cells in human pancreatic adenocarcinoma-bearing mice: Randomized study

AimsTo investigate the possible acute toxicities and pathological changes associated with intravenous, intraperitoneal, or intratumoral injection of natural killer (NK) cells in mice subcutaneously bearing human pancreatic adenocarcinoma (PaC). Methods100 NPG tumor-bearing mice (50/sex) were engrafted subcutaneously with human PaC BXPC-3 cells 9 days before administration. They were randomly divided into 10 groups with 5 males and 5 females in each group. Mice in Group 1 were given sodium chloride intravenously as vehicle control, and mice in Groups 2–4 human peripheral blood-derived NK cells intravenously at doses of 2 × 107, 1 × 108, and 5 × 108 cells/kg, respectively; mice in Groups 5–7 were injected with NK cells intraperitoneally at doses of 2 × 107, 1 × 108, and 5 × 108 cells/kg, respectively, and mice in Groups 8–10 with NK cells intratumorally at doses of 4 × 103, 2 × 104, and 1 × 105 cells/mm3, respectively. Each group was given a single dose; the mice were observed clinically, and body weight, food intake, blood biochemistry, and tumor volume were measured. On Day 15, the mice were euthanized for gross anatomy and histopathology. ResultsOn planned euthanasia, in Groups 2–4 no gross or microscopic pathological changes related to cells injection were found; in Groups 5–7 mice of both sexes showed a decrease in extramedullary hematopoiesis of spleen, and at the dose of 5 × 108 cells/kg, mice of both sexes showed an increase in the composition of spleen white pulp cells. In Groups 8–10, mice of both sexes at doses of 4 × 103 and 1 × 105 cells/mm3 and female mice at the dose of 2 × 104 cells/mm3 showed a decrease in extramedullary hematopoiesis of spleen, and female mice at a dose of 4 × 103 cells/mm3 and mice of both sexes at doses of ≥ 2 × 104 cells/mm3 showed an increase in the composition of spleen white pulp cells; perivascular/peribronchiolar inflammatory cell infiltration in lung and bronchus was observed in mice of both sexes at doses of ≥ 2 × 104 cells/mm3, and inflammatory cell infiltration in liver was observed in mice of both sexes at a dose of 1 × 105 cells/mm3. No other abnormal changes with toxicological significance in clinical observation, body weight, food intake, or blood biochemistry were observed in each group. ConclusionsIn our study intravenous injection appears the safest way to give NK cells to human PaC-bearing mice. Using intraperitoneal or intratumoral administration, spleen, liver, and lung were the most often affected organs, albeit with mostly mild pathological changes.

Changes in peripheral blood immune cell population in thyroid cancer patients treated with lenvatinib

This study evaluated changes in the peripheral blood immune cell population in patients with advanced thyroid cancer receiving lenvatinib treatment to confirm the immune-modulatory effect of lenvatinib. After obtaining informed consent from patients, we prospectively collected 20 ml of whole blood at 2–3 months intervals 2–4 times from each patient; peripheral blood mononuclear cells (PBMCs) were separated, and the Maxpar Direct Immune Profiling Assay was performed. A total of 10 patients were enrolled, and 31 blood samples were obtained. The median age of patients was 65 years, and all patients showed durable responses to the lenvatinib treatment. When we compared the PBMC profiles between the pre-treatment, on-treatment, and off-treatment samples, the peripheral natural killer (NK) cell proportion differed significantly. The proportion of NK cells among total live cells significantly increased from 9.3 ± 4.5 (%) in the pre-treatment samples to 20.8 ± 7.9 (%) in the on-treatment samples (P = 0.009) and decreased to 13.3 ± 3.1 (%) in the off-treatment samples (P = 0.07). There was a significant increase in the peripheral NK cell population with lenvatinib treatment in advanced thyroid cancer patients. This finding confirms the immune-modulatory effect of lenvatinib.

NK cell phenotypic profile during active TB in people living with HIV-evolution during TB treatment and implications for bacterial clearance and disease severity

Natural killer (NK) cells, key effector cells of the innate immune system, play an important role in the clearance and control of Mycobacterium tuberculosis and HIV infections. Here, we utilized peripheral blood specimens from the Improving Retreatment Success CAPRISA 011 study to characterize NK cell phenotypes during active TB in individuals with or without HIV co-infection. We further assessed the effects of TB treatment on NK cell phenotype, and characterized the effects of NK cell phenotypes during active TB on mycobacterial clearance and TB disease severity measured by the presence of lung cavitation. TB/HIV co-infection led to the expansion of functionally impaired CD56neg NK cell subset. TB treatment completion resulted in restoration of total NK cells, NK cell subset redistribution and downregulation of several NK cell activating and inhibitory receptors. Higher percentage of peripheral CD56bright cells was associated with longer time to culture conversion, while higher expression of NKp46 on CD56dim NK cells was associated with lower odds of lung cavitation in the overall cohort and the TB/HIV co-infected participants. Together these results provide a detailed description of peripheral NK cells in TB and TB/HIV co-infection and yield insights into their role in TB disease pathology.

Circulating Natural Killer Cells as Prognostic Value for Non-Small-Cell Lung Cancer Patients Treated with Immune Checkpoint Inhibitors: Correlation with Sarcopenia.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of tumors. Natural killer (NK) cells can play an important role in cancer immune surveillance. The aim of this prospective observational study was to analyze peripheral blood mononuclear cells (PBMCs) in patients with advanced non-small-cell lung cancer (NSCLC) receiving ICIs in order to identify predictive factors for better survival outcomes. Forty-seven stage IV NSCLC patients were enrolled. Patients underwent baseline (T0) and longitudinal (T1) evaluations after ICIs. Peripheral immune blood cell counts were analyzed using flow cytometry. Basal levels of CD3-CD56+ NK cells were higher in patients with controlled disease (DC) compared to progression disease (PD) patients (127 cells/µL vs. 27.8 cells/µL, p < 0.001). Lower NK cell values were independent prognostic factors for shorter overall survival (OS) (HR 0.992; 95% CI 0.987-0.997, p < 0.001) and progression-free survival (PFS) (HR 0.988; 95% CI 0.981-0.994, p < 0.001). During the longitudinal evaluation, CD3-CD56+ NK cells (138.1 cells/µL vs. 127 cells/µL, p = 0.025) and CD56bright NK cells (27.4 cells/µL vs. 18.1 cells/µL, p = 0.034) significantly increased in the DC group. Finally, lower values of CD3-CD56+ NK cells (28.3 cells/µL vs. 114.6 cells/µL, p = 0.004) and CD56dim NK cells (13.2 cells/µL vs. 89.4 cells/µL, p < 0.001) were found in sarcopenic patients compared to patients without sarcopenia. Peripheral NK cells could represent a non-invasive and useful tool to predict ICI therapy response in NSCLC patients, and the association of low NK cell levels with sarcopenia deserves even more attention in clinical evaluation.