Cytolytic Natural Killer Cells Research Articles

Brief cycling intervals incrementally increase the number of hematopoietic stem and progenitor cells in human peripheral blood.

Peripheral blood stem cell (PBSC) donation is the primary procedure used to collect hematopoietic stem and progenitor cells (HSPCs) for hematopoietic stem cell transplantation. Single bouts of exercise transiently enrich peripheral blood with HSPCs and cytolytic natural killer cells (CD56dim), which are important in preventing post-transplant complications. To provide a rationale to investigate the utility of exercise in a PBSC donation setting (≈3h), this study aimed to establish whether interval cycling increased peripheral blood HSPC and CD56dim concentrations to a greater degree than continuous cycling. In a randomised crossover study design, eleven males (mean ± SD: age 25 ± 7years) undertook bouts of moderate intensity continuous exercise [MICE, 30min, 65%-70% maximum heart rate (HRmax)], high-volume high intensity interval exercise (HV-HIIE, 4 × 4min, 80%-85% HRmax) and low-volume HIIE (LV-HIIE, 4 × 2min, 90%-95% HRmax). The cumulative impact of each interval on circulating HSPC (CD34+CD45dimSSClow) and CD56dim concentrations (cells/µL), and the bone marrow homing potential of HSPCs (expression of CXCR-4 and VLA-4) were determined. There was an increase in HSPC concentration after two intervals of LV-HIIE (Rest: 1.84 ± 1.55 vs. Interval 2: 2.94 ± 1.34, P = 0.01) and three intervals of HV-HIIE only (Rest: 2.05 ± 0.86 vs. Interval 3: 2.51 ± 1.05, P = 0.04). The concentration of all leukocyte subsets increased after each trial, with this greatest for CD56dim NK cells, and in HIIE vs. MICE (LV-HIIE: 4.77 ± 2.82, HV-HIIE: 4.65 ± 2.06, MICE: 2.44 ± 0.77, P < 0.0001). These patterns were observed for concentration, not frequency of CXCR-4+ and VLA-4+ HSPCs, which was unaltered. There was a marginal decrease in VLA-4, but not CXCR-4 expression on exercise-mobilised HSPCs after all trials (P < 0.0001). The results of the present study indicate that HIIE caused a more marked increase in HSPC and CD56dim NK cell concentrations than MICE, with mobilised HSPCs maintaining their bone marrow homing phenotype. LV-HIIE evoked an increase in HSPC concentration after just 2 × 2-minute intervals. The feasibility and clinical utility of interval cycling in a PBSC donation context should therefore be evaluated.

IL-33 supplementation improves uterine artery resistance and maternal hypertension in response to placental ischemia.

Preeclampsia (PE), a leading cause of maternal/fetal morbidity and mortality, is a hypertensive pregnancy disorder with end-organ damage that manifests after 20 wk of gestation. PE is characterized by chronic immune activation and endothelial dysfunction. Clinical studies report reduced IL-33 signaling in PE. We use the Reduced Uterine Perfusion Pressure (RUPP) rat model, which mimics many PE characteristics including reduced IL-33, to identify mechanisms mediating PE pathophysiology. We hypothesized that IL-33 supplementation would improve blood pressure (BP), inflammation, and oxidative stress (ROS) during placental ischemia. We implanted intraperitoneal mini-osmotic pumps infusing recombinant rat IL-33 (1 µg/kg/day) into normal pregnant (NP) and RUPP rats from gestation day 14 to 19. We found that IL-33 supplementation in RUPP rats reduces maternal blood pressure and improves the uterine artery resistance index (UARI). In addition to physiological improvements, we found decreased circulating and placental cytolytic Natural Killer cells (cNKs) and decreased circulating, placental, and renal TH17s in IL-33-treated RUPP rats. cNK cell cytotoxic activity also decreased in IL-33-supplemented RUPP rats. Furthermore, renal ROS and placental preproendothelin-1 (PPET-1) decreased in RUPP rats treated with IL-33. These findings demonstrate a role for IL-33 in controlling vascular function and maternal BP during pregnancy by decreasing inflammation, renal ROS, and PPET-1 expression. These data suggest that IL-33 may have therapeutic potential in managing PE.NEW & NOTEWORTHY Though decreased IL-33 signaling has been clinically associated with PE, the mechanisms linking this signaling pathway to overall disease pathophysiology are not well understood. This study provides compelling evidence that mechanistically links reduced IL-33 with the inflammatory response and vascular dysfunction observed in response to placental ischemia, such as in PE. Data presented in this study submit the IL-33 signaling pathway as a possible therapeutic target for the treatment of PE.

AT1-AA Is Produced in Offspring in Response to Placental Ischemia and Is Lowered by B-Cell Depletion Without Compromising Overall Offspring Health.

Preeclampsia, new-onset hypertension during pregnancy alongside other organ dysfunction, is the leading cause of mortality for the mother and low birth weight for the baby. Low birth weight contributes to high risk of cardiovascular disorders later in life. Women with preeclampsia have activated B cells producing agonistic autoantibodies to AT1-AA (angiotensin II type I receptor). We hypothesize that rituximab, a B cell-depleting chemotherapeutic, will deplete maternal B cells in reduced uterine perfusion pressure (RUPP) rats without worsening the effect of placental ischemia on pup growth and survival. To test this hypothesis, the RUPP procedure was performed, and rituximab was continuously infused via miniosmotic pump. Maternal blood and tissues were collected. A separate group of dams were allowed to deliver, pup weights were recorded, and at 4 months of age, tissues were collected from offspring. Immune cells were measured via flow cytometry, and AT1-AA was quantified using a contraction bioassay. Blood pressure increased in RUPP rats and was normalized with rituximab treatment. RUPP offspring also had increased circulating B cells, cytolytic natural killer cells, and increased circulating AT1-AA, which were normalized with maternal rituximab treatment. This is the first study to analyze the AT1-AA in RUPP offspring, which was normalized with rituximab. Our findings indicate that perinatal rituximab lowers maternal mean arterial pressure in RUPP rats and improves birth weight, circulating AT1-AA, and circulating natural killer cells, indicating that rituximab improves adverse fetal outcomes in response to placental ischemia.

Progesterone-induced blocking factor blockade causes hypertension in pregnant rats.

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension in mid-late gestation and can include multi-organ dysfunction with or without proteinuria. It affects 5%-7% of all pregnancies in the U.S., making PE a major contributor to maternal and fetal morbidity and mortality. Currently, there is no cure for this pregnancy complication except for early delivery of the placenta and fetus. Moreover, the therapeutic options to treat PE are very limited. One potential trigger for the development of PE is progesterone deficiency-induced imbalance between T Helper 1(Th1)/Th2 cells, an increase in cytolytic natural killer (NK) cells and inflammatory cytokines that in turn leads to endothelial dysfunction, intrauterine growth restriction (IUGR) and hypertension. Importantly, progesterone signals the synthesis of progesterone-induced blocking factor (PIBF) which has anti-inflammatory effects and could promote the regulation of inflammation balance during pregnancy. However, the role of progesterone and PIBF in the pathophysiology of PE is still not fully understood. Thus, this current study was designed to test the hypothesis that inhibition of PIBF causes signs of PE in pregnant Sprague Dawley rats. In order to address our hypothesis, rabbit anti-PIBF IgG (0.25, low dose-LD or 0.50mg/mL, high dose-HD) was administered intraperitoneally on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats. On GD 18, carotid catheters were inserted and on GD 19 mean blood pressure (MAP) and samples were collected for further analysis. MAP in normal pregnant rats (NP) rats (n=7) was 99±3mmHg, which increased to 116±2 mmHg in NP+ anti-PIBF LD (n=10) and 113±4mmHg in NP+ anti-PIBF HD (n=4), p<0.05. Plasma TNF-alpha levels were 35±8 pg/mL in NP rats and increased to 84±21 pg/mL in NP+ Anti-PIBF HD (n=4), p<0.05. Plasma IL-4 and IL-10 levels were 22±5 and 25+6 pg/mL in NP (n=5), which decreased to 6±1 and 8±1 pg/mL in NP+ Anti-PIBF LD (n=6, p<0.05) and 16±4 and 15±5 pg/mL in NP+ Anti-PIBF HD (n=4). Circulating total NK cells were 67±11 % gatein NP rats (n=3), which decreased to 28±7% gate in NP+ Anti-PIBF LD and 45±6% gate in NP+ Anti-PIBF HD. Cytolytic NK cells were increased in NP+ Anti-PIBF HD, p<0.05. Moreover, circulating NO levels were significantly decreased while renal cortex PPET-1 levels increased NP+ Anti-PIBF HD. Our study demonstrates that PIBF blockade causes hypertension, inflammation and signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signalling pathways during a healthy pregnancy.

Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

In people with HIV (PWH), the post-antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling and Tcell receptor (TCR) sequencing and examine proliferation to assess how ART impacts Tcell homeostasis. In PWH on long-term ART, lymphocyte frequencies and phenotypes are mostly stable. By contrast, broad phenotypic changes in natural killer (NK) cells, γδ Tcells, B cells, and CD4+ and CD8+ Tcells are observed in the post-ART window. Whereas CD8+ Tcells mostly restore, memory CD4+ T subsets and cytolytic NK cells show incomplete restoration 1.4 years post ART. Surprisingly, the hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1%-11% of all CD4+ Tcells) remain stable post ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+ Tcell absolute number, phenotypes, and function. The slow restoration of host immunity post ART also has implications for the design of ART interruption studies.

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Cellular Immune Composition in Multiple Myeloma Patients Associated with Variable Humoral Responses to Sars-Cov-2 Vaccination

Background: Multiple Myeloma (MM) patients are immune-deficient and mount suboptimal immune responses to SARS-CoV-2 mRNA vaccine, making them more susceptible to severe COVID-19 manifestations. We have previously described that MM patients undergoing certain therapies have compromised humoral immune responses even after receiving multiple doses of the SARS-CoV-2 mRNA vaccine (Aleman, et al. Cancer Cell 2022). However, the mechanisms underlying the suboptimal antibody responses observed in these patients are not understood. Emerging research suggests that specific cell populations within the immune milieu, such as Follicular helper (TFH) CD4+ T cells, help B cells mount a robust serological response (Cui, et al. 2021). In addition, Natural Killer (NK) cells have been known to play a protective role in SARS-CoV-2 infection (Fuentes-Villalobos, et al. 2022). We report here on the cellular immune composition highly associated with suboptimal humoral immune responses in MM patients. Methods: We longitudinally followed 31 MM patients with variable serological responses after receiving SARS-CoV-2 vaccination, as determined by quantification of anti-spike IgG antibody titers, and compared to 13 age matched healthy donors. We categorized patients based on their production of anti-spike IgG post full vaccination of two doses. Of our selected cohort, 14 patients had failed to produce detectable anti-spike IgG (MM sub-optimal), whereas the other 17 MM patients had detectable anti-spike IgG (MM within detectable range). We analyzed samples collected post second dose and post third dose of COVID-19 vaccine using high dimensional spectral flow cytometry to elucidate immune phenotypic differences. Data and sample collection for this study was approved by the institutional review board (IRB) and follows the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice (IRB: GCO#: 16-00791 (MM patients) & GCO #: 20-03374 (PARIS). Results: We compared spike binding antibody responses pre and post third vaccine dose in our MM and control cohorts. While the third dose of mRNA vaccine resulted in a robust increase in anti-spike IgG for the majority of patients, the serological response in MM patients remained significantly lower (p<0.05) as compared to healthy controls (Figure 1A). The sub-optimal MM patient group had the lowest serological response, with 28% (4/14) of the patients showing no detectable anti-spike IgG levels post third booster vaccination. MM patients with sub-optimal responses did not have an increased incidence of lymphopenia or difference of absolute B cell and T cell counts. Further examination, however, revealed noticeable differences in TFH and NK cell populations. Sub-optimal MM patients showed a significantly lower prevalence of PD1+CXCR5+ TFH cells (p<0.01), as compared to MM patients within detectable serological range both pre and post third vaccination (Figure 1B). PD1+CXCR5+ TFH cells are associated with higher levels of anti-spike IgG neutralizing antibodies (Cui, et al. 2021). In addition, sub-optimal MM patients showed significantly fewer NK cells (p<0.05) when compared to MM patients within detectable serological range and healthy donors (Figure 1B). NK cell subset analysis in the sub-optimal MM patient group revealed a high prevalence of immature NK cell subsets (CD16-/dimCD56hi) (p<0.01), whereas MM patients within serological range and healthy donors showed a dominant mature cytolytic NK cell subset (CD16hiCD56dim). Half of the sub-optimal MM patients were on anti-CD38 monoclonal antibody therapy (7/14) compared to those mounting efficient responses (11%, 2/17), which we have previously described as affecting anti-spike IgG production and depleting NK populations. Conclusion: We describe here that significantly reduced numbers of CD4+ TFH and NK cells is associated with sub-optimal serological responses to SARS-COV-2 mRNA vaccination in MM patients. The distinct phenotype observed in our sub-optimal MM patient cohort could be a product of their pre-existing immune composition, the nature of their disease progression, or in response to ongoing therapies. Our data suggests that longitudinal immunophenotyping may identify vulnerable MM patients that may need additional vaccinations or prophylactic intervention, such as monoclonal antibody therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune cell differences between patients in different stages of monoclonal plasma cell disorders.

8065 Background: Multiple myeloma (MM) is an incurable plasma cell malignancy. Precursor conditions include monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM). Immune surveillance is paramount for keeping the malignancy in check, whereas a dysfunctional immune system is suspected to promote disease progression. Methods: We performed flow cytometric immune panel analyses including T-cell, B-cell, natural killer (NK) cell and dendritic cell (DC) subsets in MGUS, SMM, and MM patients between 2018-2021. Immune panels were analyzed on fresh peripheral blood samples drawn at diagnosis. The patient population were MGUS (n=28), SMM (n=19) and MM (n=94). Samples from MGUS and SMM patients were combined into a single group (asymptomatic) and were compared to MM patients (symptomatic). Lymphocytes were identified by CD45+ expression resolving against side-scattered light on a bivariate plot. B cell and NK cell antigen expression profiles were assessed from this lymphocyte population. T cell subsets were further determined by CD3+. The percent gated for each marker was summarized by cohort using the appropriate descriptive statistics and compared using the Mann-Whitney U exact test. Analyses were performed in SAS v9.4 (Cary, NC) using a two-sided α=0.05. Results: Compared to MGUS/SMM patients, MM patients had a significant lower proportion of naive B cells, NK cells, and cytolytic NK cells, and a significant higher proportion of cytolytic T cells at diagnosis (Table). Consistent with prior studies, we demonstrated an increase in exhaustion markers. There was a trend toward a higher population of CD8+ exhausted T cells in MM patients compared to MGUS/SMM patients. Since the follow up for this study was short, survival data is not mature. Conclusions: This study demonstrates the immune imbalance that occurs between myeloma precursor conditions and MM. There is a shift in certain cell subsets in the immune microenvironment that alters tumor immunosurveillance and tumor cell killing favoring disease progression. In data not shown, we are currently analyzing the bone marrow microenvironment and its relation to disease progression. Follow up studies could assess if therapeutic intervention could reverse the imbalance, restore homeostatic equilibrium with the goal of controlling disease long-term. [Table: see text]

Manipulating CD4+ T Cell Pathways to Prevent Preeclampsia.

Preeclampsia (PreE) is a placental disorder characterized by hypertension (HTN), proteinuria, and oxidative stress. Individuals with PreE and their children are at an increased risk of serious short- and long-term complications, such as cardiovascular disease, end-organ failure, HTN, neurodevelopmental disorders, and more. Currently, delivery is the only cure for PreE, which remains a leading cause of morbidity and mortality among pregnant individuals and neonates. There is evidence that an imbalance favoring a pro-inflammatory CD4+ T cell milieu is associated with the inadequate spiral artery remodeling and subsequent oxidative stress that prime PreE’s clinical symptoms. Immunomodulatory therapies targeting CD4+ T cell mechanisms have been investigated for other immune-mediated inflammatory diseases, and the application of these prevention tactics to PreE is promising, as we review here. These immunomodulatory therapies may, among other things, decrease tumor necrosis factor alpha (TNF-α), cytolytic natural killer cells, reduce pro-inflammatory cytokine production [e.g. interleukin (IL)-17 and IL-6], stimulate regulatory T cells (Tregs), inhibit type 1 and 17 T helper cells, prevent inappropriate dendritic cell maturation, and induce anti-inflammatory cytokine action [e.g. IL-10, Interferon gamma (IFN-γ)]. We review therapies including neutralizing monoclonal antibodies against TNF-α, IL-17, IL-6, and CD28; statins; 17-hydroxyprogesterone caproate, a synthetic hormone; adoptive exogenous Treg therapy; and endothelin-1 pathway inhibitors. Rebalancing the maternal inflammatory milieu may allow for proper spiral artery invasion, placentation, and maternal tolerance of foreign fetal/paternal antigens, thereby combatting early PreE pathogenesis.

TIMP1 and TIMP2 Downregulate TGFβ Induced Decidual-like Phenotype in Natural Killer Cells.

Simple SummaryCancer patients are characterized by NK cells with altered surface markers, such as CD56 brightness, CD9, CD49a (pro-angiogenic) and PD-1, and TIM-3 (exhaustion), that favor immune escape. Transforming growth factor-beta (TGFβ) is a major tumor-derived cytokine that favors cancer growth and supports pro-angiogenic activities in NK cells by inducing pro-angiogenic molecules. TIMP-1 and TIMP-2 play a crucial role in extracellular matrix (ECM) regulation, wound healing, pregnancy and cancer, and there is increasing evidence that they are immune-modulatory. We found that recombinant TIMP-1 and -2 can partially contrast the induction of pro-tumor/pro-angiogenic decidual-like polarization of NK cells by TGFβ.Natural Killer (NK) cells have been found to be anergic, exhausted and pro-angiogenic in cancers. NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like-phenotype, together with decreased levels of NKG2D activation marker, increased levels of TIM-3 exhaustion marker, similar to cancer-associated NK cells. Tissue inhibitors of metalloproteases (TIMPs) exert dual roles in cancer. The role of TIMPs in modulating immune cells is a very novel concept, and the present is the first report studying their ability to contrast TGFβ action on NK cells. Here, we investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16−, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Treatment with recombinant TIMP-1 or TIMP-2 showed a trend, although not statistically significant, to decrease CD49a+ and TIM-3+ expression and increase NKG2D in peripheral blood NK cells exposed to conditioned media from colon cancer cell lines. Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. Given the heterogeneity of released factors within the TME, it is clear that TGFβ stimulation represents a model to prove TIMP’s new properties, but it cannot be envisaged as a soloist NK cell polarizing agent. Therefore, further studies from the scientific community will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic–therapeutic roles.

Nilotinib/Interferon-α Combination Rapidly Enhances Leukaemia-Associated Antigen-Specific Cytotoxic T-Lymphocyte Immune Responses, Limits Natural Killer Cell Maturation and Triggers B Cell Remodelling

▪Aim: We investigated the immune effects of nilotinib (NIL) combined with pegylated-interferon-α2b (IFN) in a subgroup of newly diagnosed chronic phase Chronic Myeloid Leukemia (CML) patients within the ALLG CML11 PINNACLE trial. NIL was given for the first 3 months (mo), followed by combination with IFN for 21 mo (from mo 4 - 24), then NIL alone from 25 mo.Methods: We assessed 12 patients serially from diagnosis, 2, 3, 5, 7, 10, 16 and 28 mo, including samples at 1, 8 and 15 days post-IFN commencement (median of 8 samples per patient). Multi-parameter flow cytometry was used to characterise effector phenotype and function of innate immune Natural Killer (NK) cells, Regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), CD4+/CD8+ T cells and B cells. Functional cytotoxic T-lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were assessed by interferon-γ (IFN-γ)/tumour-necrosis factor-α (TNF-α) FLUROSPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A*0201 specific peptides in HLA-A*0201+ patients.Results: All patients in this cohort had BCR-ABL1 <10% at 3 mo following NIL alone, 91% had optimal BCR-ABL1 responses (<1% and <0.1% at 6 and 12 mo respectively) with NIL/IFN. Cytolytic CD3-CD56dimCD16br NK cells increased after NIL (median; diagnosis 20.6% vs 2 mo 38.6%, p=0.002), the phenotype was more mature (CD57+) with increased CD161, NKp46, and NKp30 receptors. In contrast, the absolute number of CD56dimCD16br NK cells reduced as early as 8 days post-IFN, was lowest at 7 mo (151 cells/μl) on combination treatment vs 2 mo NIL alone (543 cells/μl, p=0.01). NK maturation status also decreased post-IFN and was subsequently restored upon IFN cessation (Table 1). A trend toward increasing immunoregulatory CD56brCD16-/dim NK cells was observed post-IFN (10 mo; 58 cells/μl) vs 2 mo NIL alone (32 cells/μl). NKp30/46 expression increased further on combination therapy, other NK receptors including CD161, KIR2DL2/DL3/DS2 and KIR2DL5 expression and functional degranulation response were unaffected by IFN. Programmed death-1 receptor expression, indicative of immune activation, increased on NK cells post-IFN, but was unchanged in T cells, B cells and monocytes.Low CD62L which is associated with impaired immune function was observed on CD4+/CD8+ T cells at diagnosis. CD62L surface expression increased from 2 mo NIL and remained increased with NIL/IFN combination. CD62L was highest at 28 mo. Naïve/memory T cell analysis showed decreasing CD8+ but not CD4+ effector memory (EM) cells on combination therapy (10 mo; 13.7%) vs diagnosis (23.3%, p=0.04) and 2 mo NIL (23.9%) with no change in central memory (CM). Functional IFN-γ and/or TNF-α LAA-CTL responses were observed in 83% of patients, with peak responses 15 days post-IFN. PRAME-CTLs (predominantly IFN-γ driven) were the most abundant and detected in all responders, with BMI-1- and PR3-CTLs in 50% of responders.CD3-CD19+ B cell numbers remained unchanged however, Transitional B cells (CD24++CD38++) the first cells which migrate to the peripheral blood, rapidly increased as early as 15 days post-IFN (283 cells/μl) vs diagnosis (70 cells/μl, p=0.001) and 2 mo NIL (61 cells/μl, p=0.008). CD27-IgD+ Naïve B cells demonstrated a similar increased pattern of expression. In contrast, CD27+IgD+ non class switched and CD20+CD38dim class switched memory B cells decreased following combination therapy.Suppressor Treg numbers decreased post-NIL vs diagnosis, and increased with IFN. Treg displayed a shift from EM phenotype to CM on NIL, and remained unchanged on combination therapy. T cell immunoreceptor TIGIT+ Tregs representing a highly activated/suppressive phenotype were unchanged at all time points. Predominantly granulocytic (Gr)-MDSC (CD66b+CD15+) were observed at diagnosis, reducing early in patients on NIL alone and remaining low throughout the study period.Conclusion: This first immunological report of NIL/IFN combination in newly diagnosed CML patients identifies rapid and divergent immunomodulatory effects encompassing both the innate and adaptive immune system. IFN alters NK cell phenotype, without affecting effector function, increases specific early B cell subsets, is associated with enhanced LAA-CTL responses and reduces suppressive Gr-MDSC. Correlation of patient immune responses and clinical outcomes is ongoing. [Display omitted] DisclosuresWhite:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Yeung:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Hughes:BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Yong:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Research Funding; Novartis: Honoraria, Research Funding.

Inhibitory axes impacting on the activity and fate of Innate Lymphoid Cells

In neoplastic patients, an effective immune response ideally should be achieved by the coordinated action of different immune cells with tumor-suppressive functions. These include the more cytolytic members of the Innate Lymphoid Cells (ILCs) family represented by the Natural Killer (NK) cells, whose activities in cancer patients, however, can be hampered by several inhibitory signals. These are generated by membrane-bound and soluble molecules that, interacting with specific inhibitory receptors, create inhibitory axes impacting the NK cell differentiation and effector functions. These breaks, which now represent major immunotherapeutic targets, may be sensitive to interferon (IFN)-γ, whose source, in vivo, is represented by different cell types including the NK and ILC1. Since also ILCs can express receptors of the inhibitory axes like PD-1 and TIGIT, their therapeutic blockade might further amplify the IFN-γ release that, as an unwanted side effect, would promote the onset of NK cell-resistant tumor variants (NKRTV) expressing ligands involved in inhibitory axes. These variants might also arise from the activity of other cytokines such as IL-27, which can increase the expression of HLA class I and PD-Ls in different cell types, including tumor cells. Besides the amplification of membrane-bound inhibitory axes, tumors can reduce the number of infiltrating cytolytic ILCs, promote the recruitment of poorly cytolytic NK cell subsets, and manipulate to their advantage the infiltrating immune cells, which acquire tumor-promoting activities. This occurs thanks to the production of soluble factors including TGF-β1 and IL-18 that, alone or in combination, modify the activating and chemokine receptor repertoire of NK cells, and induce the ILCs differentiation towards cells ineffective in fighting cancer or, even worse, with tumor-promoting functions.The present review aims to present and discuss major inhibitory axes impacting on ILCs functions, migration, and differentiation with a major focus on tumor context.

Effects of Bacillus firmus e volumine ex muris cellulae 6x on cytolytic activity of natural killer cells in vitro

Natural killer (NK) cells are among the first in defense of the innate immune system by eliminating a variety of abnormal or stressed cells such as cancer cells or virus-infected cells. Individuals who exhibit low cytolytic NK cell activity are believed to be at higher risk of viral infection, tumorigenesis, and various other diseases of the immune system. Therefore, restoration of impaired NK cell function might be an essential step in immunostimulatory therapy of immunocompromised patients. Bacillus firmus is a non-pathogenic gram-positive bacterium of the environment, which possesses various immunomodulatory properties in vitro and in vivo. This retrospective study reports on the effect of B. firmus on the activity of NK cells in vitro. Basal cytolytic NK cell activity against tumor cells among peripheral blood mononuclear cells (PBMCs) of routine patients was determined in a standardized NK cell cytotoxicity assay. The impact of cultivation of PBMCs with B. firmus preparation Bacillus firmus e volumine ex muris cellulae (Bacillus firmus (evc)) 6x on tumor cell killing by NK cells was monitored in relation to basal NK cell activity. This study showed that stimulation of PBMCs with Bacillus firmus (evc) 6x in vitro led to a significant increase in NK cell function. Substantial improvement in cytolytic NK cell activity (more than 1.3-fold of basal activity) was much more pronounced for patients with compromised NK cell function. Due to its immunostimulatory mode of action, Bacillus firmus (evc) may be of particular importance in therapy of patients with NK cell deficiency.

Interferon γ neutralization reduces blood pressure, uterine artery resistance index, and placental oxidative stress in placental ischemic rats.

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction, and increased cytolytic natural killer cells (cNKs), which secrete interferon γ (IFNγ). However, the precise role of IFNγ in contributing to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that neutralization of IFNγ in RUPPs will decrease placental reactive oxygen species (ROS) and improve vascular function resulting in decreased MAP and improved fetal growth. On gestation day (GD) 14, the RUPP procedure was performed and on GDs 15 and 18, a subset of normal pregnant rats (NP) and RUPP rats were injected with 10 μg/kg of an anti-rat IFNγ monoclonal antibody. On GD 18, uterine artery resistance index (UARI) was measured via Doppler ultrasound and on GD 19, mean arterial pressure (MAP) was measured, animals were euthanized, and blood and tissues were collected for analysis. Increased MAP was observed in RUPP rats compared with NP and was reduced in RUPP + anti-IFNγ. Placental ROS was also increased in RUPP rats compared with NP rats and was normalized in RUPP + anti-IFNγ. Fetal and placental weights were reduced in RUPP rats, but were not improved following anti-IFNγ treatment. However, UARI was elevated in RUPP compared with NP rats and was reduced in RUPP + anti-IFNγ. In conclusion, we observed that IFNγ neutralization reduced MAP, UARI, and placental ROS in RUPP recipients. These data suggest that IFNγ is a potential mechanism by which cNKs contribute to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes in PE.

Tumor Necrosis Factor-alpha Blockade Improves Uterine Artery Resistance, Maternal Blood Pressure, and Fetal Growth in Placental Ischemic Rats.

We recently reported that adoptive transfer of cytolytic Natural Killer cells (cNKs) from the Reduced Uterine Perfusion Pressure (RUPP) rat induces a preeclampsia (PE)-like phenotype in pregnant rats, accompanied by increased TNF-α. The purpose of this study was to investigate a role for increased TNF-α to induce oxidative stress (ROS), decrease nitric oxide (NO) bioavailability, and induce vascular dysfunction as mechanisms of hypertension (HTN) and intrauterine growth restriction (IUGR) in RUPPs. Pregnant Sprague Dawley rats underwent the RUPP or a Sham procedure on gestation day (GD) 14. On GDs 15 and 18, a subset of Sham and RUPP rats received i.p.injections of vehicle or 0.4mg/kg of Etanercept (ETA), a soluble TNF-α receptor (n=10/group). On GD18, Uterine Artery Resistance Index (UARI) was measured, and on GD19, mean arterial pressure (MAP), fetal and placental weights were measured, and blood and tissues were processed for analysis. TNF-α blockade normalized the elevated MAP observed RUPP. Additionally, both fetal and placental weights were decreased in RUPP compared to Sham, and were normalized in RUPP+ETA. Placental ROS was also increased in RUPP rats compared to Sham, and remained elevated in RUPP+ETA. Compared to Sham, UARI was elevated in RUPPs while plasma total nitrate was reduced, and these were normalized in ETA treated RUPPs. In conclusion, TNF-α blockade in RUPPs reduced MAP and UARI, improved fetal growth, and increased NO bioavailability. These data suggest that TNF-α regulation of NO bioavailability is a potential mechanism that contributes to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes and fetal growth.

Progesterone and PIBF: new insights into treatment options for preeclampsia

We have shown Preeclampsia (PE) to be a progesterone deficient state of pregnancy associated with increased TH1 lymphocytes, cytolytic natural killer (NK) cells, inflammatory cytokine, vasoactive pathways resulting in endothelial function and hypertension. Healthy normal pregnancy (NP) is associated with elevations in progesterone and TH2 lymphocytes, and uterine NK cells favoring immunotolerance toward the fetus. Importantly, NP lymphocytes express progesterone receptors, which stimulate Progesterone Induced Blocking Factor (PIBF) upon binding to progesterone, thus PIBF increases during NP. Therefore we hypothesize that PIBF blockade causes a proinflammatory hypertensive phenotype similar to PE. Rabbit anti-PIBF IgG (0.50 mg/mL) was administered i.p. on gestational day 15 (GD 15) to NP rats, on GD 18 carotid catheters were inserted and on GD 19 mean arterial blood pressure (MAP) and samples were collected. MAP in NP rats (n=7) was 99+ 3 mmHg, which increased to 113+4 mmHg in NP+ anti-PIBF (n=6), p<0.05. Plasma TNF-α was 35+8 pg/mL in NP rats and increased to 84+21 pg/mL in NP+ Anti-PIBF (n=4), p<0.05. Circulating total NK cells were 67+ 11 in NP rats (n=4), which decreased to 36+4 in NP+ Anti-PIBF, while cytolytic NK cells were 0.6 + 0.2 in NP and increased to 3.0+1 in NP+ Anti-PIBF, p<0.05. Circulating nitric oxide was 44+ 11 µM in NP rats (n=5), which decreased to 21+1 µM in NP+ Anti-PIBF (n=6), p<0.05, while, renal cortex preproendothelin-1 increased 15 fold in NP+ Anti-PIBF (n=6) compared to NP rats (n=5). In order to determine if PIBF supplementation improves a PE phenotype during pregnancy, PIBF (2.0 µg/mL) was administered i.p. on GD 15 to the reduced uterine perfusion pressure (RUPP) a rat model of PE and compared to control RUPP and NP rats. On GD19, MAP and samples were collected. MAP in NP rats (n=11) was 100+ 2 mmHg, 105 + 3 in NP+PIBF (n=8), 122+ 1 in RUPP rats (n=10), which improved to 110+2 mmHg in RUPP+PIBF rats (n=11), p<0.05. PIBF lowered circulating and placental cytolytic NK cells in RUPPs from 15+6, 2.4+1% gate to 3+2, 0.4+0.1% in RUPP+PIBF(p<0.05, n=4-8). Moreover PIBF supplementation increased circulating IL-4 and TH2 to 40±8 pg/mL, 2+0.6 % gate in RUPP+PIBF (n=4-8 p<0.05) compared to9 ±2 pg/mL, 0.5+ 0.1 % gate in RUPP. Importantly, vasoactive pathways preproendothelin-1 and nitric oxide were normalized in RUPP+PIBF rats compared to RUPP rats, p<0.05. Collectively, these data demonstrate an important role for PIBF to control the inflammatory milieu thereby normalizing vasoactive pathways and maintaining healthy blood pressures during pregnancy.

THE IMPORTANCE OF B2 CELLS IN THE HYPERTENSION AND PATHOPHYSIOLOGY OF PLACENTAL ISCHEMIA DURING PREGNANCY

Objective: Preeclampsia (PE), is characterized by placental ischemia, inflammation, hypertension and IUGR. Antibodies activating the angiotensin II type I receptor (AT1-AA) activate cytolytic Natural Killer cells (NK) and cause clinical features of PE. The Reduced uterine perfusion pressure (RUPP) pregnant rat model of PE has elevated AT1-AA, NK cells and blood pressure. B cell depletion or AT1-AA epitope binding protein (‘n7AAc’) improved hypertension and AT1-AA activity and NK cells in RUPP rats. One unanswered question is the importance of B2 cells vs B1 cells in the production of the AT1-AA and thus the pathology of PE. Design and method: To test this hypothesis, splenic B Cells and B2 cells were isolated from RUPP rats on Gestation Day (GD) 19 and transferred into NP rats on GD12. Blood pressure (MAP) and cNKs were measured by flow cytometry on GD19. To test the role of AT1-AA in the hypertension, ‘n7AAc’ was administered to a group of recipient rats on GD14. Results: MAP increased in NP+RUPP B cells to 117 ± 1 mmHg, n = 10, and to 111 mmHg ± 3, n = 8, in NP+RUPP B2 cells, compared to NP rats, n = 10 (p &lt; 0.001). ‘n7AAc’ attenuated hypertension in response to total RUPP B cells (99 mmHg ± 6, n = 5)(p &lt; 0.01) and in response to RUPP B2 s (100 mmHg ± 5, p = 0.08). Circulating and placental cNKs were elevated with total RUPP B cells and RUPP B2 cells compared to NP. Circulating cNKs were elevated in NP+RUPPBcells (6 ± 2.5, n = 5)(p &lt; 0.01) and in NP+RUPP B2 cells (8 ± 3.33, n = 4)(p &lt; 0.01) compared to NP (0.67 ± 0.33, n = 10). Placental cNKs were elevated in NP+RUPPBcells (2.32 ± 0.73, n = 6)(p &lt; 0.01) and in NP+RUPP B2 cells (1.33 ± 0.24, n = 3)(p &lt; 0.01) compared to NP (0.2250 ± 0.133, n = 10). Conclusions: We conclude that RUPP total B Cells and RUPP B2 cells cause hypertension and NK cell activation via secretion of the AT1-AA during pregnancy. However, our studies do not rule out the importance of B1 cells in hypertension and AT1-AA production during PE or RUPP. Future studies from our laboratory will further examine B1 cells in the pathology of PE.

Dissecting the Role of BET Bromodomain Proteins BRD2 and BRD4 in Human NK Cell Function.

Natural killer (NK) cells are innate lymphocytes that play a pivotal role in the immune surveillance and elimination of transformed or virally infected cells. Using a chemo-genetic approach, we identify BET bromodomain containing proteins BRD2 and BRD4 as central regulators of NK cell functions, including direct cytokine secretion, NK cell contact-dependent inflammatory cytokine secretion from monocytes as well as NK cell cytolytic functions. We show that both BRD2 and BRD4 control inflammatory cytokine production in NK cells isolated from healthy volunteers and from rheumatoid arthritis patients. In contrast, knockdown of BRD4 but not of BRD2 impairs NK cell cytolytic responses, suggesting BRD4 as critical regulator of NK cell mediated tumor cell elimination. This is supported by pharmacological targeting where the first-generation pan-BET bromodomain inhibitor JQ1(+) displays anti-inflammatory effects and inhibit tumor cell eradication, while the novel bivalent BET bromodomain inhibitor AZD5153, which shows differential activity towards BET family members, does not. Given the important role of both cytokine-mediated inflammatory microenvironment and cytolytic NK cell activities in immune-oncology therapies, our findings present a compelling argument for further clinical investigation.

HIV-infected macrophages resist efficient NK cell-mediated killing while preserving inflammatory cytokine responses

Natural killer (NK) cells are innate cytolytic effectors that target HIV-infected CD4+ Tcells. In conjunction with antibodies recognizing the HIV envelope, NK cells also eliminate HIV-infected targets through antibody-dependent cellular cytotoxicity (ADCC). However, how these NK cell functions impact infected macrophages is less understood. We show that HIV-infected macrophages resist NK cell-mediated killing. Compared with HIV-infected CD4+ Tcells, initial innate NK cell interactions with HIV-infected macrophages skew the response toward cytokine production, rather than release of cytolytic contents, causing inefficient elimination of infected macrophages. Studies with chimeric antigen receptor (CAR) Tcells demonstrate that the viral envelope is equally accessible on CD4+ Tcells and macrophages. Nonetheless, ADCC against macrophages is muted compared with ADCC against CD4+ Tcells. Thus, HIV-infected macrophages employ mechanisms to evade immediate cytolytic NK cell function while preserving inflammatory cytokine responses. These findings emphasize the importance of eliminating infected macrophages for HIV cure efforts.

Progesterone-induced blocking factor improves blood pressure, inflammation, and pup weight in response to reduced uterine perfusion pressure (RUPP).

Preeclampsia (PE) is characterized by new-onset hypertension in association with elevated natural killer (NK) cells and inflammatory cytokines, which are likely culprits for decreased fetal weight during PE pregnancies. As progesterone increases during normal pregnancy, it stimulates progesterone-induced blocking factor (PIBF). PIBF has been shown to decrease inflammation and cytolytic NK cells, both of which are increased during PE. We hypothesized that PIBF reduces inflammation as a mechanism to improve hypertension in the preclinical reduced uterine perfusion pressure (RUPP) rat model of PE. PIBF (2.0 µg/mL) was administered intraperitoneally on gestational day 15 to either RUPP or normal pregnant (NP) rats. On day 18, carotid catheters were inserted. Mean arterial blood pressure (MAP) and samples were collected on day 19. MAP in NP rats (n = 11) was 100 ± 2 mmHg and 105 ± 3 mmHg in NP+PIBF rats (n = 8) and 122 ± 1 mmHg in RUPP rats (n = 10), which improved to 110 ± 2 mmHg in RUPP+PIBF rats (n = 11), P < 0.05. Pup weight was 2.4 ± 0.1 g in NP, 2.5 ± 0.1 g in NP+PIBF, 1.9 ± 0.1 g in RUPP, and improved to 2.1 ± 0.1 g in RUPP+PIBF rats. Circulating and placental cytolytic NK cells, IL-17, and IL-6 were significantly reduced while IL-4 and T helper (TH) 2 cells were significantly increased in RUPP rats after PIBF administration. Importantly, vasoactive pathways preproendothelin-1, nitric oxide, and soluble fms-Like tyrosine Kinase-1 (sFlt-1) were normalized in RUPP+PIBF rats compared with RUPP rats, P < 0.05. Our findings suggest that PIBF normalized IL-4/TH2 cells, which was associated with improved inflammation, fetal growth restriction, and blood pressure in the RUPP rat model of PE.