Mature Natural Killer Cells Research Articles

Should the Treatment of Patients with Repeated Embryo Implantation Failure Be Adapted as a Function of the Endometrial Cytokine Profile? A Single-Center Experience.

Repeated embryo implantation failures (RIF) is a source of distress and frustration for patients and clinicians alike. Today's approaches for treating RIF are largely empirical and have limited effectiveness. The main causes of RIF are poor endometrial receptivity and poor-quality embryos. Recent studies have suggested the involvement of immune dysregulation due to an imbalance between T-helper (Th) 1 and Th2 cytokines; this opens up perspectives for treating women with RIF and increasing the implantation rate. We conducted an interventional, longitudinal, prospective cohort study of the impact of correcting the cytokine imbalance on the clinical pregnancy rate in women with RIF. Seventy-seven women with RIF underwent an endometrial biopsy during the implantation window. The cytokine profile was evaluated by studying the activation and maturation of uterine natural killer (uNK) cells, the IL-15/Fn-14 mRNA ratio (a biomarker of uNK activation/maturation), and the IL-18/TWEAK mRNA ratio (a marker of angiogenesis and the Th1/Th2 balance). Personalized treatment was initiated for women with an abnormal endometrial cytokine profile (hyper-activation or hypo-activation). We documented the clinical pregnancy rate after subsequent embryo transfers. In total, 72.7% (56/77) of patients had an abnormal endometrial cytokine profile (hyper-activation in 68.8% (n = 53) and hypo-activation in 3.9% (n = 3). After treatment (or not) as a function of the endometrial profile, the overall clinical pregnancy rate was 30.2%. Our results indicated a potential positive effect of appropriate treatment on the ongoing pregnancy rate in women with RIF, despite the small number of cases analyzed. The results must now be validated in randomized studies with larger numbers of well-characterized patients. By applying a previously published decision tree, this treatment approach could be implemented in clinics worldwide.

Newborn bacille Calmette-Guérin vaccination induces robust infant interferon-γ-expressing natural killer cell responses to mycobacteria

ObjectivesThe bacille Calmette-Guérin (BCG) vaccine is usually administered at birth to protect against severe forms of tuberculosis in children. BCG also confers some protection against other infections, possibly mediated by innate immune training. We investigated whether newborn BCG vaccination modulates myeloid and natural killer (NK) cell responses to mycobacteria. MethodsBCG vaccination was either administered at birth or delayed to 6 or 10 weeks of age in 130 South African infants. Whole blood was stimulated with BCG and clusters of differentiation (CD)4+ T, myeloid, and NK cell responses were measured by flow cytometry; the levels of secreted cytokines were measured by a multiplex bead array. ResultsNewborn BCG vaccination was associated with significantly higher frequencies of BCG-reactive, cytokine-expressing CD4+ T cells, and interferon (IFN)-γ-expressing NK cells than in unvaccinated infants but no differences in cytokine-expressing CD33+ myeloid cells were observed. The induction of BCG-reactive IFN-γ-expressing NK cells was not associated with the markers of NK cell maturation, differentiation, or cytokine receptor expression. BCG-reactive NK cell responses correlated directly with the levels of secreted interleukin (IL)-2 and IFN-γ and the innate pro-inflammatory cytokines IL-6, IL-1β, and tumor necrosis factor (TNF) in BCG-vaccinated infants only. ConclusionWe showed that BCG-reactive IFN-γ-expressing NK cells are strongly induced by BCG vaccination in infants and are likely amplified through bystander cytokines.

Natural Killer Cell Epigenetic Reprogramming in Tumors and Potential for Cancer Immunotherapy

Natural killer (NK) cells are critical members of the innate lymphoid cell population and have a pivotal role in cancer eradication. NK cell maturation, development and function are tightly regulated by epigenetic modifications, which can also be recruited for cancer propagation and immune escape. NK cells have the potential to be activated against tumors through several epigenetic regulators. Given that epigenetic changes are inducible and reversible, focusing on aberrant epigenetic regulations recruited by tumor cells provides a tremendous opportunity for cancer treatment. This review presents a comprehensive picture of NK cell normal epigenetic regulation and cancer-driven epigenetic modifications. From our perspective, a better understanding of epigenetic regulators that can edit and revise NK cells' activity is a promising avenue for NK cell-based therapy in cancer management.

Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses

Dendritic cell (DC)-based cancer vaccines are a form of immunotherapy that activates the innate and adaptive immune systems to combat cancers. Neutrophils contribute to cancer biology and have the potential to be exploited by immunotherapeutic platforms to enhance anti-tumor immune responses. We previously showed that DC vaccines elicit the expansion of mouse interferon (IFN)γ-producing mature natural killer (NK) cells to elevate anti-tumor responses. Here, we demonstrate the rapid recruitment of neutrophils to the draining lymph nodes of DC-vaccinated mice. This was accompanied by an increase in the total number of NK cells producing IFNγ and expressing CD107a, a marker of degranulation that demonstrates NK cell functional activity. Furthermore, the depletion of neutrophils in DC-immunized mice resulted in decreased numbers of NK cells in draining lymph nodes compared to the controls. Interestingly, the increased number of IFNγ- and CD107a-expressing NK cells in DC-immunized mice was not detected in mice depleted of neutrophils. Further investigations showed that DC vaccines induced IFNγ- and TNFα-producing CD8+ T cells that also expressed CD107a, but depletion of neutrophils did not have any impact on the CD8+ T cell population. Our findings suggest that neutrophil-mediated anti-tumor immunity induced by a DC vaccine platform could be targeted to provide innovative strategies to enhance its clinical efficacy.

Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion,NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.

IRF2 is required for development and functional maturation of human NK cells.

Natural killer (NK) cells are cytotoxic and cytokine-producing lymphocytes that play an important role in the first line of defense against malignant or virus-infected cells. A better understanding of the transcriptional regulation of human NK cell differentiation is crucial to improve the efficacy of NK cell-mediated immunotherapy for cancer treatment. Here, we studied the role of the transcription factor interferon regulatory factor (IRF) 2 in human NK cell differentiation by stable knockdown or overexpression in cord blood hematopoietic stem cells and investigated its effect on development and function of the NK cell progeny. IRF2 overexpression had limited effects in these processes, indicating that endogenous IRF2 expression levels are sufficient. However, IRF2 knockdown greatly reduced the cell numbers of all early differentiation stages, resulting in decimated NK cell numbers. This was not caused by increased apoptosis, but by decreased proliferation. Expression of IRF2 is also required for functional maturation of NK cells, as the remaining NK cells after silencing of IRF2 had a less mature phenotype and showed decreased cytotoxic potential, as well as a greatly reduced cytokine secretion. Thus, IRF2 plays an important role during development and functional maturation of human NK cells.

Haploidentical Hematopoietic Stem Cell Transplant Plus Add-Back NK Cells in Pediatric High-Risk ALL

Introduction: Natural killer (NK) lymphocytes are cells of innate immunity that protect against tumor and infections. NK cells recognize self and non-self HLA class I ligands, are regulated by a balance between activator and inhibitory signals through surface receptors, and can interact with HLA class I molecules on target cells. Killer-cell Immunoglobulin-like Receptors (KIRs) are inhibitory molecules expressed on NK cells and belong to the immunoglobulin superfamily. KIRs function to "educate" NK cells in effective defense against unhealthy self/non-self cells and tolerance against healthy self cells. NKs are thought to play a key role in preventing early relapse of hematologic malignancies, virus infection and graft versus host disease (GvHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT).In B-/T-cell alpha/beta depleted haplo-HSCT, donor-versus-recipient allo-reactive NK cells can exert an efficient graft versus leukemia (GvL) effect, mediated by inhibitory KIRs where the HLA ligand is missing on recipient cells. Donor NK cells after haplo-HSCT are "immature" and not effective as the mature NKs. These immature NK cells are CD56bright, are derived from CD34+ stem cells, and mature to a CD56dim phenotype. The mature CD56dim NK cells are characterized by acquisition of KIRs, CD16 and CD57 in mature stages, which have functional significance in targeting and medicating their cytolytic activity. Based on these considerations, we performed haplo-HSCT with donor NK cell add-back to provide mature, functional immunological surveillance against tumor (MRD) and virus reactivation, which can frequently occur in manipulated haplo-HSCT for pediatric acute lymphoblastic leukemia (ALL). Methods: We evaluated the alloreactive effect of donor-derived NK cells in three pediatric high-risk ALL who underwent B-/T-cell alpha/beta depleted haplo-HSCT followed by donor NK infusion. Patients' characteristics are summarized in the Table. All 3 patients were male; median age was 13 months. Patients 1 and 2 had infant ALL, and patient 3 had refractory-relapsed (r/r) ALL. All patients received a median of 3 lines chemotherapy. Patient 1 received a prior allo-HSCT. Patients 1 and 2 received chemo-based conditioning regimens consisting of thyotepa, and fludarabine; In addition, patient1received treosulfan, and patient 2 received busulfan. Treosulfan was given to patient 1, due to less toxicity profile given prior allo-HSCT. Patient 3 received Total Body Irradiation (TBI), thyotepa, and fludarabine. All patients received rituximab and letermovirfor EBV and CMV prophylaxis, respectively, for all patients. Mother was Donor for all patients. Median CD34+, CD19+, CD3+-alpha/beta cell dose was 8.8, 0.05, 0.41 x 106/kg, respectively. NK cell allo-reactivity was tested by evaluating KIR genotyping and KIR/KIR-ligand mismatch in graft-versus-host direction. All three patients present KIR/KIR-ligand mismatch. Results: Engraftment was day +13, +14 and +19 for ANC, and +14, +18 and +17 for PLT (for both >20K and >50K), for patients 1, 2 and 3, respectively. NK cell number were monitored on day +7, +14, +21 and +28 (data pending). Patients received NK cell infusion on day 30 +/- 2 days. No infusion reactions were observed. MedianNK infused were 256x106/kg. None of the patients developed CMV, EBV or ADV reactivation. Patient 2 developed Grade 2 acute GvHD (skin) treated with steroids. No Grade GvHD 3-4 was observed. Median follow up was 58 days. All patients had full donor chimerism at day 30 ±2. All patients are currently in hematological CR; patient 1 converted to positive MRD at nine months from transplant. Conclusion: Alloreactive NK cell add-back infusion after B-/T-cell alpha/beta depleted haplo-HSCT could represent an effective option to improve transplant outcome and prevent virus reactivation. These NK cells may improve efficacy by enhancing GvL and could contribute to maintain the "disease control" through immunologic surveillance in high-risk ALL. Indeed, NK killing, which is higher in mature NK cells, depends on acquisition of sequential phenotypic maturation. Thus, infusion of mature donor NK cells may help to maintain control of MRD and virus reactivation through immunologic surveillance. Although our preliminary observations seem to be promising, this hypothesis should be confirmed in clinical trial of a larger number of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Method for Efficient cGMP Production of Natural Killer Cells from Clonal Master Induced Pluripotent Stem Cells for Next Generation, Off-the-Shelf Cancer Immunotherapy

Natural killer (NK) cells are innate immune cells that play a key role in in tumor immune surveillance. NK cells are self-tolerant to healthy cells and can kill tumor cells in both hematological and solid malignancies. Recent advances allow for the derivation of immune cells, including NK cells, from human induced pluripotent stem cells that can be utilized for cancer immunotherapy. High doses of NK cells (approx. 0.5-5 x 109 cells per dose) and multiple doses are both safe and likely necessary for clinical efficacy. Manufacturing large number of NK cells from a clonal master iPSC line provides a promising strategy to enable next generation, off-the-shelf, affordable, cancer immunotherapies. We have developed a novel process to produce high purity, clinical scale NK cells efficiently and consistently. This method does not require cell sorting or the usage of murine-derived stromal cells. Briefly, hematopoietic progenitor cells are derived using an improved spin embryoid body (EB) method using defined cytokines including bone morphogenic protein-4 (BMP-4), stem cell factor (SCF), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase 3 (FLT-3) etc. The hematopoietic progenitor cells are subsequently differentiated into mature NK cells using a second set of defined cytokines such as IL-3, IL-7, IL-15, SCF etc. Using this novel method, fully functioning NK cells could be expanded more than 8,000-fold to enable us to produce 1 x 1012 NK cells starting from 1 x 106 undifferentiated iPSCs. This cell production scale can supply >200 clinical doses from one cGMP manufacturing campaign. NK cells produced using this method are CD56-positive and display the typical phenotype of NK cell activating receptors including NKG2D, NKp44, NKp46, DNAM-1. Importantly, these NK cells demonstrate better anti-tumor activities and produce higher levels of IFNγ and TNFα compared to peripheral blood-derived NK cells isolated from healthy donors. Moreover, this protocol has been adapted and optimized for clinical scale manufacturing of NK cells from a genetically engineered iPSC cell line bearing a knock-out of CISH, a key intracellular checkpoint of NK cell activation. CISH KO NK cells produced using this optimized method show significantly better anti-tumor activities against multiple liquid and solid tumor cell lines (Figure 1), including K562, Raji (B cell lymphoma), Daudi (B cell lymphoma), SUP-B15 (B cell lymphoma), MOLT-4 (T cell lymphoma), SKOV-3 (ovarian cancer), OVCAR-4 (ovarian cancer), HCC-827 (lung cancer), ZR-75 (breast cancer), and BT-474 (breast cancer), in standard cytotoxicity assays compared with unmodified NK cells. Overall, this novel cell production strategy paves the way for clinical trials using higher doses of iPSC-derived NK cells with increased potency, thus enabling next-generation CAR-NK cell-based immunotherapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow Microenvironment Changes in Myelodysplastic Neoplasms and Its Relationship with Clonal Hematopoiesis and Disease Progression

Background: Immune dysregulation and somatic gene mutations are known prognostic factors in myelodysplastic neoplasms (MDS). During the evolution of the disease, a decrease in the immune surveillance is observed, with impaired cytotoxicity and a decrease in mature natural killer cells (NK), among other changes. Also, over expression of inflammatory cytokines, plays a role in the immune environment. The interaction between immune populations and the malignant clone is not entirely understood, especially in the bone marrow. The aims of this study were to characterize bone marrow (BM) immune populations, especially NK cells, and its correlation with the malignant clone. Methods: We prospectively collected 70 BM samples. Fifty MDS without RS or 5q deletion, 11 cytopenia of unknown significance (CUS) and 9 healthy donors (HD). The aims were to analyze NK cells, defined as (CD45+CD3-CD19-) and differentiating an immature population (CD56brigthCD16−NKG2A/CD94+KIR+/−) from a mature one (CD56dim CD16+NKG2A/CD94−KIR+). Their different receptors: activators (NKp46, NKp30, NKG2C, NKG2D, NKp44, DNAM) and inhibitors (TIGIT, NKG2A, Irp60, and PD1), as well as clonal cells expression of their ligands (HLA-ABC, MICA-B, CD155, PD-L1). Finally, KIR haplotype was analyzed, by NGS. Other populations included were myeloid-derived suppressor cells (MDSC), differentiating granulocytic (Gr-MDSC: CD11b+CD33+HLA-DR-CD15+CD14-) and monocytic (Mo-MDSC:CD11b+CD33+ HLA-DRlow/CD15+ CD14+). The analysis was performed, by flow cytometry, on BM fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Finally, cytokines concentrations were stablished with the commercial kit ProcartaPlexTM Multiplex immunoassay. Results: A total of 70 subjects (median age of 77 years old and 45% female) were included (Figure 1). Although, total NK was similar among the three groups [MDS 0.17x109/L ICUS 0.07x109/L, HD 0.20x109/L, p= 0.1204], we observed differences between NK subpopulations. An increase of mature NK cells CD56dimCD16+ in CUS (76.16%) compared with MDS (11.58%) and HD (13.67%) [p= 0.0005] was observed. Regarding NK activating receptors, DNAM receptor was highly expressed on MDS (96.05%) and HD (90.98%), compared to CUS (67.79%) [p= 0.007], and the expression of KIR2DS4 was decreased in MDS (28.47%) compared to HD (79.4%) [p= 0.005], as well as NKp44 on MDS (0.19%) compared with HD (0.34%) [p= 0.03]. Interestingly, no expression of KIR2DS4 or NKp44 were observed on CUS. Regarding inhibitor receptors, Irp60 was increased on MDS (95.52%) compared to CUS (85.07%) and HD (80.2%) [p= 0.036] (Figure 2). Inhibitory KIR haplotype AA was observed on 13/47 MDS (27.56%). Finally, no differences were observed between Gr-MDSC [MDS 0.51%, ICUS 0.13%, HD 0.33%, p=0.35] or Mo-MDSC [MDS 1.03%, ICUS 0.25%, HD 0.7%, p=0.28] expression, among groups. Eighty-four percent (42/50) of MDS presented mutations at diagnosis, with a median number of 3. Interestingly, 63.6% (7/11) of CUS presented mutations, 2 DMT3A (VAF < 5%), 1 TET2 (VAF 40%), 1 IDH1 and 1 SF3B1 (VAF 33%), among others. Within subjects presenting mutations, we observed differences on the expression of NKG2A, showing an increase on those with ≥3 mutations (37.23%) vs <3 mutations (35.3%) [p=0.02]. Moreover, differences were found on KIR2DL1 [≥3 mutations 6.66% vs <3 mutations 15.61%, p=0.05]. On the 7 clonal cytopenia of unknown significance (CCUS), no differences were found on the expression of NK receptors compared to idiopathic cytopenia of unknown significance (ICUS). Cytokine analysis on BM of 39 MDS, showed increased levels of IL-10 in HR compared to LR [2 pg/ml vs. 1 pg/ml, p=0.04], including 16 (53%) subjects with very high concentrations (> 40 pg/ml). In addition, we analyzed NK cells of 14 MDS subjects who had a second BM sample, one year after diagnosis. We documented an increase in CD244 NK population [15.23% follow-up vs 3.61% diagnosis, p=0.05]. Conclusions: Our study suggests the presence of immunosuppressive profile of NK cells on MDS, with gain of expression of NKG2A and higher expression of IL10 on high risk MDS. Interestingly, 63.6% CUS subjects were CCUS and showed increase cytotoxic profile. The increase of CD244 expression, at one year control, may be related to an exhausted environment leading to immunosuppression and progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trial in Progress: AB-101, an Allogeneic, Non-Engineered, Cord Blood-Derived Natural Killer (NK) Cell Therapy, As Monotherapy or in Combination with Rituximab in Patients with Relapsed or Refractory Non-Hodgkin Lymphoma

Background: Natural Killer (NK) cells are known to eliminate tumors directly or via antibody-dependent cellular cytotoxicity (ADCC) which is dependent on the maturation, activation and FcγRIIIA (CD16) phenotype of NK cells. Many cancer patients have low NK cell number and dysfunctional innate immunity, which has been correlated with outcomes to monoclonal antibody (mAb) therapy. AB-101 is an allogeneic, non-genetically modified cord blood-derived NK cell therapy, developed to potentially enhance patients' innate anti-tumor activity in combination with approved oncology products with known ADCC mechanism of action. AB-101 has been optimized for combination with mAbs through the novel AlloNK™ proprietary large scale manufacturing process utilizing donor cord blood units. The cord blood unit is selected for key attributes, including an immunoglobulin-like receptor B (KIR-B) haplotype and the natural high-affinity variant of CD16 (158V/V polymorphism). The AlloNK™ process results in an activated and mature NK cell product with increased cell surface expression of CD16, NKG2D, NKp30/44/46, and DNAM-1, without any requirement for genetic engineering. Additionally, the process generates sufficient NK cells to treat hundreds to thousands of patients from a single cord blood unit and produces a consistently active NK cell product, with little donor-to-donor variability. The combination of AB-101 and rituximab has demonstrated strong cytotoxic activity in preclinical studies; the combination led to improved survival over rituximab or NK cells alone in xenograft mouse models. As a cryopreserved, "off-the-shelf", and infusion-ready product, AB-101 is being evaluated for the treatment of hematologic malignancies in combination with standard of care antibody therapies. Study Design, Treatment and Methods: AB-101-01 is a first in human, Phase 1/2, multi-center, open-label study to evaluate the safety and anti-tumor activity of AB-101 as monotherapy and in combination with rituximab in patients with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) of B-cell origin or CLL. This study has 3 parts: Phase 1, 1b, and 2. Phase 1 will evaluate 2 dose levels as monotherapy or in combination with rituximab using the 3+3 dose escalation method. Phase 1b will enroll up to 45 patients in combination with rituximab to determine the recommended phase 2 dose (RP2D). Phase 2 will enroll an additional 15 patients at the RP2D. AB-101 will be given at 1 or 4 billion cells per dose. All enrolled patients will receive at least 1 treatment cycle of 4 doses of AB-101, administered once weekly. Prior to receiving the first dose of AB-101, patients will receive 3 consecutive days of lymphodepleting chemotherapy consisting of fludarabine (30 mg/m2/day) and cyclophosphamide (250 or 500 mg/m2/day). After each AB-101 infusion, patients will receive interleukin-2 (IL-2) at 1×106 IU/m2/dose or 6×106 IU/dose to potentiate NK cell activation and persistence. If given, 375 mg/m2 of rituximab is dosed on the second day of lymphodepletion and with the second and fourth dose of AB-101. Patients who have a complete or partial response or maintain stable disease with the first cycle of the combination may receive 3 additional cycles such that cycle 2 is given with lymphodepletion and cycles 3 and 4 do not include lymphodepletion. Eligible patients must have a diagnosis of B-cell NHL or CLL and must have received at least 2 prior lines of therapy, including an anti-CD20 antibody therapy. Treatment with prior CD19 CAR T-cell therapy is permitted. Patients must be at least 18 years of age, have ECOG PS of 0 or 1, and have measurable disease per Lugano 2014 at the time of enrollment. The primary objectives of each phase are as follows: in Phase 1, the evaluation of the safety of 1 and 4 billion cells/dose of AB-101 as monotherapy or in combination with rituximab through the incidence of AEs and DLTs; in Phase 1b, determination of the RP2D based on safety and antitumor response to AB-101 with rituximab; and in Phase 2, further characterization of the efficacy profile. The study is currently enrolling at 10 U.S. centers. (ClinicalTrials.gov Identifier: NCT04673617)

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

OMIP-086: Full spectrum flow cytometry for high-dimensional immunophenotyping of mouse innate lymphoid cells.

This 25-parameter, 22-color full spectrum flow cytometry panel was designed and optimized for the comprehensive enumeration and functional characterization of innate lymphoid cell (ILC) subsets in mouse tissues. The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterization of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimization of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin, and pro-ILC activating cytokines.

A Sampling of Highlights from the Literature.

Macrophages can be reprogrammed to promote antitumor responses (by Noah Smith via Wikimedia Commons).Multiple factors contribute to the suppression that occurs in the tumor microenvironment (TME) and this could potentially be alleviated through various mechanisms. Chen et al. find that high potassium in the TME inhibits antitumor responses from tumor-associated macrophages (TAM) via alteration of their metabolism. Blocking this metabolic switch enables TAMs to mount effective antitumor responses. Chryplewicz et al. find that TAMs in glioma are reprogrammed to a proinflammatory phenotype, along with other antitumor remodeling of the TME and tumor vasculature, after treatment with VEGF inhibitors and imipramine (tricyclic anti-depressant). These studies provide insight into multiple mechanisms of TAM suppression and potential targets that can be used to boost antitumor responses.Chen S, …, Wang D. Cell Metab 2022 September 13. DOI:10.1016/j.cmet.2022.08.016.Chryplewicz A, …, Hanahan D. Cancer Cell 2022 September 15;40:1111–27.E9.Dietary intake can affect microbiota and immunity (by the NCI via Wikimedia Commons).Dietary intake can affect antitumor immunity and response to immunotherapy. In a prospective study, Simpson et al. evaluated dietary patterns and microbial signatures of different patient populations and how these factors related to response to neoadjuvant immune checkpoint inhibition. Distinct differences between response groups were observed not only in microbial composition but also in immune-related adverse events. Specific diets, as well as certain microbial compositions, were associated with better treatment outcomes. The data highlight the crossroads of cancer-intrinsic and -extrinsic factors with patient lifestyle, and how these factors together can shape responses to treatment.Simpson RC, …, Long GV. Nat Med 2022 September 12. DOI:10.1038/s41591-022-01965-2.Immunotherapy can overcome resistance to HER2-targeted therapy (from IrinaPav via Wikimedia Commons).HER2-specific antibodies such as trastuzumab are used to treat HER2+ breast cancer, but not all patients respond and some that do ultimately relapse. Using orthogonal approaches, including analysis of tumor tissue and transcriptomic data from trastuzumab-treated patients with HER2+ breast cancer and a fully humanized immunocompetent model of HER2+ breast cancer, Rivas et al. find that a population of TGFβ-activated cancer-associated fibroblasts (CAF) and low IL2 activity associate with trastuzumab resistance. Targeting IL2 to fibroblast activation protein-α (FAP)–expressing cells restores tumor responsiveness to trastuzumab by enhancing NK cell–mediated antibody-dependent cytotoxicity, highlighting the therapeutic potential of targeting stromal components in the tumor microenvironment.Rivas EI, …, Calon A. Nat Commun 2022 September 9;13:5310.A Treg subpopulation of CAR T cells drives therapeutic resistance (from NIH via Flickr).Using single-cell profiling methods, two groups have identified subpopulations of CD19-targeting CAR T cells with T regulatory (Treg)–cell phenotypes associated with poor response in patients with B-cell lymphoma. Good et al. find a higher proportion of circulating CD4+Helios– Treg CAR T cells in patients with progressive disease who received axicabtagene ciloleucel (axi-cel), highlighting their potential as a biomarker prognostic for progression at day 7 post infusion. Haradhvala et al. similarly identify CAR+ Treg cells in blood and infusion products for patients nonresponsive to either axi-cel or tisagenlecleucel, and validate their role in mouse models of CAR T-cell treatment. The studies provide new insight into therapeutic resistance to CAR T-cell therapy.Good Z, …, Mackall CL. Nat Med 2022 September 12;28:1860–71.Haradhvala NJ, …, Maus MV. Nat Med 2022 September 12;28:1848–59.Covalent K-Ras(G12C) inhibitor provides a new target for immunotherapy (from Rawpixel).MHC class I presentation of tumor-specific peptides derived from intracellular proteins provides a target for anticancer therapy. Zhang et al. have identified a new class of therapeutically targetable MHC class I epitopes: covalently modified intracellular proteins. K-Ras(G12C) covalently conjugated to the K-Ras(G12C)-specific inhibitor ARS1620 can undergo antigen processing and presentation on MHC class I. ARS1620-specific antibodies can recognize MHC class I–restricted ARS1620-peptides. Incorporating such an antibody into a bispecific T-cell engager (BiTE) induces T-cell responses against K-Ras(G12C) tumor cells, suggesting a new strategy for cancer immunotherapy.Zhang Z, …, Craik CS. Cancer Cell 2022 September 12;40:1060–69.e7.NK cells are key component of the antitumor immune response (from NIAID via Wikimedia Commons).Understanding the factors that regulate the maturation and function of natural killer (NK) cells is important for understanding the antitumor immune response. Imianowski et al. show that the transcriptional regulator BACH2 is expressed in developing and mature NK cells and is a negative regulator of NK-cell maturation and function, keeping NK cells quiescent under conditions of IL15 stimulation. In mice lacking BACH2 in NK cells, the NK cells show increased cytotoxicity and enhanced ability to limit metastasis to the lungs in two preclinical models. The data provide new avenues for potential therapeutic modulation of NK cell–mediated antitumor immunity.Imianowski CJ, …, Roychoudhuri R. J Exp Med 2022 September 30;219:e20211476.

Cadmium impairs the development of natural killer cells and bidirectionally modifies their capacity for cytotoxicity

Cadmium (Cd) is a highly toxic heavy metal in the environment. The aim of this study was to investigate the impact of Cd on natural killer (NK) cells. C57BL/6 mice were treated with 10 ppm Cd via drinking water for 3 months, and the development of NK cells in the bone marrow (BM) and the cytotoxicity of mature NK (mNK) cells in the peripheral immune organs were evaluated thereafter; the impact of Cd on the cytotoxicity of mNK cells from human peripheral blood mononuclear cells (PBMC) was also investigated. Whereas Cd treatment impaired the differentiation of NK progenitors in the BM, Cd treatment activated the JAK3/STAT5 signaling to drive the proliferation of mNK cells and thereby lead to a compensation increase of mNK cells in the peripheral immune organs of mice. Additionally, Cd treatment bidirectionally regulated the cytotoxicity of mouse mNK cells to differential tumor cells, dependent on the levels of Fas expression in the tumor cells; mechanically, Cd treatment activated the JAK3/STAT5 signaling to promote the expression of FasL in mNK cells to increase their cytotoxicity, while Cd treatment reduced the expression of granzyme B in mNK cells to impair their cytotoxicity in the peripheral immune organs of mice. Likewise, in vitro assays indicated that Cd treatment also activated the JAK3/STAT5 signaling to increase the expression of FasL, whereas Cd treatment reduced the expression of granzyme B in human mNK cells. Thus Cd treatment impaired the development of NK cells in the BM and bidirectionally regulated the cytotoxicity of mNK cells in the peripheral immune organs, which may extend our current understanding for the immunotoxicity of Cd.

The effects of B-cell-activating factor on the population size, maturation and function of murine natural killer cells.

The role of B-cell-activating factor (BAFF) in B-lymphocyte biology has been comprehensively studied, but its contributions to innate immunity remain unclear. Natural killer (NK) cells form the first line of defense against viruses and tumors, and have been shown to be defective in patients with systemic lupus erythematosus (SLE). The link between BAFF and NK cells in the development and progression of SLE remains unstudied. By assessing NK cell numbers in wild-type (WT), BAFF-/- (BAFF deficient), BAFF-R-/- (BAFF receptor deficient), TACI-/- (transmembrane activator and calcium modulator and cyclophilin ligand interactor deficient), BCMA-/- (B-cell maturation antigen deficient) and BAFF transgenic (Tg) mice, we observed that BAFF signaling through BAFF-R was essential for sustaining NK cell numbers in the spleen. However, according to the cell surface expression of CD27 and CD11b on NK cells, we found that BAFF was dispensable for NK cell maturation. Ex vivo and in vivo models showed that NK cells from BAFF-/- and BAFF Tg mice produced interferon-γ and killed tumor cells at a level similar to that in WT mice. Finally, we established that NK cells do not express receptors that interact with BAFF in the steady state or in the BAFF Tg mouse model of SLE. Our findings demonstrate that BAFF has an indirect effect on NK cell homeostasis and no effect on NK cell function.

INFLAMMATORY CYTOKINES PERTURB NK CELL FUNCTION AND GENE SIGNATURE IN CHRONIC MYELOID LEUKEMIA

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent anti-cancer immunotherapeutic. NK cells'mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in CML patients. The molecular drivers of this impairment during leukemic progression remain unclear. Given the established role of inflammatory cytokines (namely IL1β, IL-6, GM-CSF, TNFα) in driving myeloid malignancies, we aimed to define their effect on NK cell phenotype and function using a unique pre-clinical chimeric mouse model of CML. We hypothesize that leukemic cytokines impede NK cell maturation and cytotoxicity and shift NK cells toward a pro-tumor cytokine-producing phenotype. We establish a chimeric mouse model of CML by transplanting bone marrow BCR-ABL+ CD45.2 cells into sub-lethally irradiated CD45.1 C57BL/6 wild-type mice. For controls, mice are transplanted with non-mutant cells. Such chimeras represent a robust model to study the exposure of non-transformed host cells to leukemia. At the endpoint, splenic, blood, and bone marrow NK cells are harvested and profiled by flow cytometry, RT-qPCR, scRNA-seq, or used for degranulation assay. Blood samples are collected to obtain serum. Consistent with clinical observations, NK cells are severely reduced in numbers and frequencies and display an immature phenotype and diminished expression of activating receptors (Nkp46, Ly49D) while upregulating inhibitory molecules (Lag-3, TIGIT, NKG2A) during leukemia. Moreover, CML-exposed NK cells have a higher proliferation rate and a greater cell death indicating perturbed homeostasis. We revealed a preferential loss of terminally mature CD27−CD11b+ NK subset in CML mice, suggesting a functional shift from cytotoxic phenotype toward the immature cytokine-producing. In agreement, CML-exposed NK cells show impaired cytotoxicity as measured by target-specific degranulation ex vivo . Next, we mapped the transcriptional landscape of CML-exposed NK cells using single-cell RNA-sequencing. We confirmed decreased gene expression of NK maturation and cytotoxicity markers ( Itgam, Cx3cr1, Prf1, Gzma ) and upregulation of inhibitory molecules ( Tigit, Lag-3, Pdcd1lg1/2) and proteins involved in the cell cycle and senescence ( Mki67, Ccna, Ccnb ). Importantly, CML-exposed NK cells had significantly increased mRNA levels of inflammatory cytokines including IL1β, TNF, GM-CSF; cytokine receptors Il6st, Il-2Ra; and negative regulators of STAT signaling SOCS1/2/3 and CIS. Furthermore, IL-6-STAT3 and IL1β/TNF-signaling gene signatures were enriched in CML-exposed NK cells – an effect that is likely to be triggered by inflammatory cytokines. Thus, we next found that peripheral blood serum from CML mice dampens healthy NK cell degranulation ex vivo , compared to serum from control mice. Further ELISA analysis revealed elevated levels of IL-6, IL-1, TNF, and GM-CSF in CML serum samples. Some of these cytokines might be secreted by functionally skewed NK cells, as suggested by gene expression analysis. Altogether, our data suggest that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing optimal targets for NK-boosting CML immunotherapies.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

Cord Blood-Derived Natural Killer Cell Exploitation in Immunotherapy Protocols: More Than a Promise?

Simple SummaryNK cell anti-tumor activity against hematological malignancies is well-established and many studies support their role in the control of solid tumor growth and metastasis generation. However, tumor microenvironment may affect NK cell function. Ongoing studies are aimed to design novel immunotherapeutic protocols to combine NK cell-based immunotherapy with other therapeutic strategies to improve the anti-tumor NK cell response. In this context, UCB is one of the main sources of both mature NK cells and of CD34+ HSPC that can generate NK cells, both in-vivo and in-vitro. UCB-derived NK cells represent a valuable tool to perform in-vitro and preclinical analyses and are already used in several clinical settings, particularly against hematological malignancies. The present review describes the characteristics of different types of UCB-derived NK cells and the in-vitro models to expand them, both for research and clinical purposes in the context of cancer immunotherapy.In the last 20 years, Natural Killer (NK) cell-based immunotherapy has become a promising approach to target various types of cancer. Indeed, NK cells play a pivotal role in the first-line defense against tumors through major histocompatibility complex-independent immunosurveillance. Their role in the control of leukemia relapse has been clearly established and, moreover, the presence of NK cells in the tumor microenvironment (TME) generally correlates with good prognosis. However, it has also been observed that, often, NK cells poorly infiltrate the tumor tissue, and, in TME, their functions may be compromised by immunosuppressive factors that contribute to the failure of anti-cancer immune response. Currently, studies are focused on the design of effective strategies to expand NK cells and enhance their cytotoxic activity, exploiting different cell sources, such as peripheral blood (PB), umbilical cord blood (UCB) and NK cell lines. Among them, UCB represents an important source of mature NK cells and CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs), as precursors of NK cells. In this review, we summarize the UCB-derived NK cell activity in the tumor context, review the different in-vitro models to expand NK cells from UCB, and discuss the importance of their exploitation in anti-tumor immunotherapy protocols.

Adaptive NK cell expansion and activity in ovarian cancer patients with cytomegalovirus infection and inflammation (258)

Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer. Objectives: Cytomegalovirus (CMV) infection has been shown to expand a subset of natural killer (NK) cells called adaptive NK cells (CD57+NKG2C+) that are long-lived, have enhanced cytotoxicity, and are thought to contribute to cancer control. CMV reactivation is associated with increased inflammation, which can impact patient outcomes. We sought to evaluate the impact of CMV exposure and inflammation, as measured by C-reactive protein (CRP), on adaptive NK cells in patients with high-grade serous ovarian cancer (HGSOC) using serum and ascites samples collected at the time of surgery. Methods: A total of 30 serum samples, collected at the time of cytoreductive surgery in women with HGSOC, were measured for CMV serostatus using an immunoassay and measured for CRP with an immunoturbidimetric method. The samples were categorized as reactive or non-reactive for CMV and low or high for CRP (≤10, >10 mg/L). Thirty serum and 23 ascites samples were evaluated by flow cytometry to quantify adaptive NK cells. A CyTOF panel of 42 antibodies was used to identify adaptive NK cells, differences in NK cell maturation and functional mediators, activation markers, a number of other immune subsets (CD8 T cells, T regulatory cells, B cells, monocytes), and proliferation signatures. Functional assays were performed to assess natural cytotoxicity and antibody-dependent cytokine cytotoxicity (ADCC) by CD1O7a degranulation and IFNy production. Data were analyzed using GraphPad Prism 8.2.0, and p-values ≤ 0.05 were considered statistically significant. Results: Patients with HGSOC with evidence of CMV exposure had increased percentages of CD57+NKG2C+ adaptive NK cells in the blood and ascites compared to those without CMV exposure (Figure 1, p=0.03). CRP blood levels were not significantly different (p=0.30). The increased percentages of adaptive NK cells, observed in the blood and ascites samples that were CMV IgG+, were not significantly altered by the presence (p=0.90) or absence (p=0.90) of CRP. Conclusions: CMV exposure influences the presence of an adaptive NK cell population in HGSOC patients. CMV expanded adaptive NK cells represent a unique NK cell population that, with their enhanced cytotoxicity and memory-like attributes, could be manipulated for future NK cell-based therapies in ovarian cancer.