Circulating NK Cells Research Articles

A phase 1 study of interleukin-15 in combination with mogamulizumab in relapsed and refractory T-cell malignancies

IntroductionRecombinant human interleukin-15 (rhIL-15) is an immunotherapeutic which enhances NK-cells to augment the antibody directed cellular cytotoxicity (ADCC) of monoclonal antibodies. Mogamulizumab is a CCR4 directed monoclonal antibodies that exerts cytotoxicity through ADCC and depletes regulatory T-cells within the tumor microenvironment. MethodsWe conducted a phase 1 clinical trial of rhIL-15 combined with mogamulizumab. Patients with relapsed or refractory adult T-cell leukemia/lymphoma (ATLL), mycosis fungoides (MF) and Sezary syndrome (SS) received fixed dose mogamulizumab combined with escalating doses of rhIL-15 to identify the maximum tolerated dose (MTD). ResultsSix patients were enrolled, 4 with ATLL and 2 with MF/SS. The most common adverse events were rash, infection and fever (67%, in all). Two patients (33%) had grade 4 acute kidney injury and 25% of cycles had grade ≥3 anemia. The MTD was dose level 1. One patient with ATLL had a partial response despite receiving only 4 cycles due to grade 4 myositis. Circulating NK cells were increased in all patients during the first cycle and a rapid reduction in tumor cells within the peripheral circulation was noted. Ex vivo assessment demonstrated increased NK cell activation and increased cell lysis in the presence of monoclonal antibodies after only 5 days. DiscussionOur small study suggests that rhIL-15 combined with mogamulizumab leads to effector NK cell activation and regulatory T-cell depletion but has an unfavorable safety profile. Future development of combinations of immunotherapy that target the microenvironment in relapsed or refractory T-cell lymphomas remain rational. NCT04185220

Proliferation capability of natural killer cells upon cytokines stimulation correlated negatively with serum lactate dehydrogenase level in coronary artery disease patients.

Natural killer (NK) cells are proposed to participate in coronary artery disease (CAD) development. However, little is known about how CAD patients' NK cells respond to different stimulatory factors in terms of proliferation capability. Twenty-nine CAD patients' peripheral blood NK cells were isolated and individually treated with IL-2, IL-12, IL-15, IL-18, IL-21, cortisone acetate, hydrocortisone, or ascorbic acid for 36 hours, followed by cell cycle analysis using flow cytometry. The ratio of S and G2/M phase cell number to total cell number was defined as a proliferation index (PrI) and used for proliferative capability indication. The results showed that these eight factors resulted in different life cycle changes in the 29 NK cell samples. Remarkably, 28 out of 29 NK cell samples showed an obvious increase in PrI upon ascorbic acid treatment. The serum lactate dehydrogenase (LDH) level of the 29 CAD patients was measured. The results showed a negative correlation between serum LDH level and the CAD patients' NK cell PrI upon stimulation of interleukins, but not the non-interleukin stimulators. Consistently, a retrospective analysis of 46 CAD patients and 32 healthy donors showed that the circulating NK cell number negatively correlated with the serum LDH level in CAD patients. Unexpectedly, addition of LDH to NK cells significantly enhanced the production of IFN-γ, IL-10 and TNF-α, suggesting a strong regulatory role on NK cell's function. Ascorbic acid could promote the proliferation of the CAD patients' NK cells; LDH serum level may function as an indicator for NK cell proliferation capability and an immune-regulatory factor.

A Pilot Study of the CD38 Antagonist Daratumumab in Patients with Metastatic Renal Cell Carcinoma or Muscle-Invasive Bladder Cancer.

We performed a pilot study of daratumumab (an mAb directed against CD38) in muscle-invasive bladder cancer (MIBC) and treatment-refractory metastatic renal cell carcinoma (mRCC). Patients with MIBC underwent baseline transurethral resection of the bladder tumor followed by four weekly doses of daratumumab prior to cystectomy. Patients with mRCC underwent baseline and sequential biopsies after eight weekly doses. The primary endpoint was safety. The secondary endpoints were pathologic complete response rate for the MIBC cohort and objective response rate and progression-free survival for the mRCC cohort. Exploratory analyses included immune monitoring and overall survival. A Bayesian sequential monitoring design for toxicity was used for excessive toxicity. In both the MIBC (n = 8) and mRCC (n = 8) cohorts, no toxicity events were encountered. In the MIBC cohort, one patient experienced pathologic complete response rate. In the mRCC cohort, no objective responses were reported, and the median progression-free survival was 1.5 months (95% confidence interval, 1.1-1.8 months). Immune monitoring found significant reductions in NK cells in circulation in both cohorts after treatment. In the tissue analysis, IHC found evidence of diminished CD38 presence in mRCC with treatment, whereas the baseline levels in MIBC were low. Treatment with daratumumab was safe. No signal of efficacy was detected in mRCC, and conclusions on the activity in MIBC were limited. Evidence of daratumumab targeting CD38 was detected in circulating immune cells and within the tumor microenvironment of mRCC and MIBC. In this prospective clinical trial of daratumumab, treatment in patients with MIBC and mRCC was safe. Limited efficacy was observed. Treatment with daratumumab resulted in CD38-expressing immune cell subsets to be targeted both in circulation and within the tumor microenvironment.

The Alteration of Circulating Invariant Natural Killer T, γδT, and Natural Killer Cells after Ischemic Stroke in Relation to Clinical Outcomes: A Prospective Case-Control Study.

The adaptive response occurs only after 7-10 days of antigen presentation. Nevertheless, the autoreactive T cells infiltrate the stroke lesion within the first 48 h. Thus, we hypothesized that the unconventional lymphocytes as invariant natural killer T cells (iNKT) and γδT cells that share immediate innate and delayed adaptive response features are involved in acute stroke pathophysiology. We assessed prospectively the quantity of circulating iNKT cells, γδT cells, and NK cells with flow cytometry in 52 subjects within three months after stroke, and we compared the results with those obtained in age-, sex-, and vascular risk factor-matched controls. We studied lymphocyte parameters regarding clinical outcomes, infarct volume, stroke-associated infection (SAI), and burden risk factors. The reduced number of circulating γδT cells and decreased percentage of the Vδ2 subset in the acute phase of stroke correlated with worse neurological status in the recovery phase. In subjects treated with thrombolysis and those who developed SAI, a lower percentage of γδT cells in the 90-day follow-up was observed. An increased percentage of iNKT cells in the acute and subacute phases of stroke was observed, and it was related to the worse clinical status. The circulating NK cells do not change temporarily or affect the outcomes after stroke. It seems that γδT cells play a long-lasting role in ischemic stroke, mainly related to the Vδ2 subset. The role of iNKT cells appears to be detrimental, especially in the acute and subacute phases of stroke. The effect of circulating NK cells on the outcome after stroke seems negligible.

Impact of urolithin A supplementation, a mitophagy activator on mitochondrial health of immune cells (MitoIMMUNE): A randomized, double-blind, placebo-controlled trial in healthy adults.

e14562 Background: Mitochondrial dysfunction and stem cell exhaustion are hallmarks of aging, driving inflammation and limiting immune function. Likewise, cancer is characterized by repression of mitochondrial dynamics in tumor-infiltrating leukocytes that fosters terminal T cell exhaustion. Yet, interventions to reliably improve immune function are still lacking. Urolithin A (UA) is a postbiotic known to improve muscle function in aged adults upon oral intake. We have recently shown that UA induces mitophagy in human CD8+ cells to induce formation of T memory stem cells, while sensitizing murine cancer to immunotherapy. Methods: In this double-blind, prospective, placebo-controlled trial (NCT05735886) we sought to characterize for the first time the effects of UA on the human immune system. Fifty healthy participants aged 45-70 years were randomized (1:1) to an intervention of single-dose (1000mg) UA or placebo per day for a study duration of 28 days. Randomization was performed based on age, gender and BMI. Exclusion criteria were prior medical conditions, BMI >35 kg/m2, smoking, and intake of prescription medication or other dietary supplements. The primary end points of the study were changes in peripheral CD3+ cells, as well as alterations in mitochondrial activity in select immune populations of the peripheral blood as assessed by flow cytometry. Results: Intake of UA was safe and well-tolerated in the intervention cohort of 25 participants. After the study period, total peripheral lymphocytes and circulating NK cells were expanded in the UA group. Performing a complete immunophenotyping via spectral flow cytometry, we found that UA elicits immune remodeling characterized by broad changes of immune surface markers and mitochondrial measurements. CD8+ cells of participants in the intervention arm displayed greater mitochondrial mass, while preferably taking on a naive phenotype and expressing more Ki67. In addition, circulating monocytes exhibited a less inflammatory signature. UA intake reduced plasma levels of several proinflammatory cytokines. Conclusions: Collectively, our study constitutes an unprecedented intervention-based assessment of immune aging that globally characterizes the effect of UA on the immune system, proposing validation in future confirmatory studies and potential combination with cancer immunotherapy. Clinical trial information: NCT05735886 .

OBX-115, an interleukin 2 (IL2)-sparing engineered tumor-infiltrating lymphocyte (TIL) cell therapy, in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma.

9515 Background: Investigational unengineered TIL cell therapy has shown promising activity in ICI-resistant metastatic melanoma but requires systemic high-dose IL2, which has well-described high-grade toxicity frequently requiring specialized management and limiting pt eligibility. OBX-115 autologous engineered TIL cell therapy does not require co-administration of IL2 due to its regulatable expression of membrane-bound IL15 (mbIL15) using the FDA-approved small-molecule drug acetazolamide (ACZ) to provide cytokine support for TIL expansion and persistence. Methods: This Phase 1 study (NCT05470283) assesses the OBX-115 regimen in pts with ICI-resistant unresectable/metastatic melanoma. OBX-115 is manufactured from the pt’s tumor tissue (surgical excision or core needle biopsy [CNB]). After lymphodepletion (cyclophosphamide, fludarabine), pts receive OBX-115 followed by ACZ once daily for up to 7 days; ACZ redosing (up to 7 days) is permitted at Wk 6 for non-responders. No systemic IL2 is administered. Peripheral blood and tumor tissue is collected for longitudinal immune profiling. Results: As of 02 Jan 2024, 9 pts (median age, 50 y) with ICI-resistant metastatic melanoma were infused with OBX-115 (median study follow up, 17 wks; range, 2–58 wks). Median lines of prior therapy was 3 (range, 1–6). OBX-115 was successfully manufactured for all pts, including from CNB tumor tissue (n = 5). The infusion product had a high proportion of CD8+ cells (≥96%) and stem-like cells (CD8+CD39-CD69-; median 76%) with very low PD-1 expression in the CD8+ population ( < 1%). Post-infusion safety included no DLTs, 3 Gr 3 nonhematologic TEAEs in 2 pts (abdominal pain, ALT elevation, syncope), and no Gr 4 nonhematologic TEAEs. ACZ was redosed and well-tolerated in 4 of 5 eligible pts. Among patients with minimum 12-wk follow-up (n = 6), ORR per RECIST v1.1 was 50% (2 CR, 1 PR, 3 SD); all responses occurred between Wk 6–18 and were ongoing, with longest response sustained > 12 mo. One pt developed new metastatic disease (liver) and progressed at Wk 24, despite continued target lesion reduction; no pt developed brain metastasis. Post-infusion ctDNA was not detectable in any of the responders at Day 14 or 42. Though OBX-115 had minimal NK cells ( < 1%), post-infusion peripheral blood and tumor biopsies showed NK cell expansion, consistent with trans-presentation of mbIL15 to circulating NK cells. Updated efficacy data on all infused pts will be presented. Conclusions: OBX-115regulatableengineeredTIL cell therapy was well-tolerated and produced consistently deepening and durable responses, indicating that OBX-115 may mediate CRs and durable clinical benefit in ICI-resistant metastatic melanoma without high-dose IL2. OBX-115 investigation continues in this and an ongoing Phase 1/2 multicenter study (NCT06060613). Clinical trial information: NCT05470283 .

#2704 Effects of high dietary salt on immunome and microbiome composition – results from a randomized clinical trial

Abstract Background and Aims Increased dietary salt intake ranks among the most prominent nutritional risk factors worldwide and has been tied to arterial hypertension and all-cause mortality. The mechanisms responsible are incompletely understood and have recently been extended to include inflammatory and microbiome-associated mechanisms. Building on our preliminary data, the present study investigates whether a moderate increase in salt intake in healthy subjects primes host physiology to a more transiently unstable state, potentially leading to changes in the microbiome-immune axis. Method We conducted a prospective, randomized, double-blinded trial to evaluate the effect of high dietary salt in healthy participants (NCT03024567), where the intervention group (n = 19) was given 6 g of salt (NaCl capsules) in addition to the habitual salt intake, resulting in an average doubling of the salt intake recommended by expert societies. A placebo group (n = 19) received gelatin capsules for 14 days. Clinical parameters, fecal samples and PBMC were collected at baseline and day 14. Shotgun metagenomic sequencing, metabolomics (stool and serum), and single-cell sequencing (Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq)) of whole PBMCs and CD4+ sorted T cells (∼150 k single cells) was performed. Results Microbiome analysis confirms previous findings showing an increased dissimilarity of microbiome composition (Jensen-Shannon Divergence) under a high-salt. Differential abundance of the microbiome's functional space, as quantified by functional gut-specific modules, picked up a salt-specific module (M00494, NatK-NatR (sodium extrusion) two-component regulatory system) in the salt intervention group. CITEseq revealed a significant reduction of circulating NK cells and an increase in classical monocytes as well as changes to several T cell subsets. Furthermore, within several immune cell subsets, we detected differently expressed genes (DEGs) solely in the salt group. We found 602 DEG in naïve conventional T cells (Tconv) and 202 DEGs in non-naïve Tconv, e.g. belonging to NF-kB and T cell receptor signaling pathways. Conclusion Our placebo-controlled study is the first to utilize different omics techniques to investigate and identify high-salt-induced changes in the microbiome and immunome in healthy individuals that may be relevant for health-to-disease transition.

Abstract 3925: Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity

Abstract Despite early diagnosis, 10-20% of breast cancer patients experience metastatic disease within 5-10 years. While it is established that breast cancer cells have the capacity to evade immune responses, the mechanisms driving this evasion remain elusive. Therefore, finding ways to enhance anti-tumor immunity is crucial to improve patient outcomes. Dickkopf-1 (DKK1) is a Wnt inhibitor whose levels correlate with poor prognosis and decreased immune infiltration in various cancers. In this study, we used mice orthotopically injected with the PyMT, EO771, and 4T1 breast cancer lines. In all models, we found elevated DKK1 serum levels, and its neutralization with an αDKK1 Ab resulted in a significant reduction in primary tumor growth but also in metastatic dissemination. To identify the source of DKK1, we examined its expression in the tumor. DKK1 was detected in stromal cells with a fibroblast-like morphology but not in cancer cells or immune infiltrates. Co-staining confirmed colocalization between DKK1 and the CAF marker αSMA. To address the role of CAF-derived DKK1, we specifically deleted DKK1 in CAFs by generating FSP1CreDkk1fl/fl and αSMACreERT2Dkk1fl/fl mice. Both models showed a significant reduction in primary tumor growth while maintaining comparable serum DKK1 levels, indicating locally produced DKK1 supports tumor growth. To understand how DKK1 promotes tumor progression, we conducted bulk RNAseq of tumor cells isolated from IgG or αDKK1 treated mice. While no changes related to cell viability or cell cycle were observed, immune response-related genes were upregulated in αDKK1-treated cells. In support of this, αDKK1 did not reduce tumor growth in NSG immune-compromised mice. To pinpoint the immune cell targeted by DKK1, we depleted T, NK cells, or macrophages in WT tumor-bearing mice treated with αDKK1. Remarkably, only NK cell depletion negated αDKK1 anti-tumor effects. In vitro assays further demonstrated that rDKK1 or the presence of CAFs reduced NK cell cytotoxicity against tumor cells while αDKK1 restored it. To assess whether DKK1 levels correlated with tumor progression and immune suppression in breast cancer patients, we analyzed DKK1 serum levels and circulating NK cells at the diagnosis of metastatic bone disease and 15-18 months after standard care. DKK1 levels were significantly elevated in patients with progressive bone disease compared to those with stable disease. Importantly, NK cells from the progressive disease cohort had reduced perforin and granzyme B expression at the follow-up visit compared to baseline, indicating reduced cytotoxicity. Furthermore, rDKK1 reduced perforin in NK cells from healthy donors. In sum, our data show DKK1 is an important regulator of breast cancer progression, and we report for the first time DKK1 directly impairs NK cell cytotoxicity, creating an immune suppressive environment resistant to treatment. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3925.

Abstract 182: Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous ovarian cancer

Abstract Objective: Here our goal was to evaluate the impact of the immunosuppressive enzyme cyclooxygenase-2 (COX-2) expression on the immune effector capacity against high-grade serous ovarian cancer (HGSOC) tumor cells through bioinformatic tools and in vitro validation. Methods: We performed an integrated bioinformatics analysis from transcriptomic data based on RNAseq (TCGA-OV, n=368; Australian cohort AOCS, n=80) and RNA microarrays (GSE26193, n=62; GSE30161, n=45). We estimated the proportion and characteristics of immune cells employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms. Survival curves and Cox-regression models were built up according to known risk factors and immune scores predicted by in-silico analysis. Afterward, we validated these results using flow cytometry and cytotoxicity assays on circulating NK cells collected from advanced HGSOC patients treated at our institution (n=5). After three days of incubation, isolated NK cell cultures were stimulated with three pulses of a selective COX-2 inhibitor (celecoxib) or MOCK, daily. An anti-CTLA-4 monoclonal antibody (ipilimumab) or MOCK was added at the third pulse. Two ovarian cancer cell lines (SKOV3 and HeyA8) were treated similarly. Next, NK cells were co-cultured with SKOV3 or HeyA8 and cytotoxicity on these cells was examined using lactate dehydrogenase (LDH) cell viability assay. Results: Transcriptomic data evidenced a non-linear relationship between COX-2 gene, PTGS2, and CTLA4 in advanced stages of HGSOC. Hence, PTGS2/CTLA4 ratio allows us to investigate how COX-2 influences changes in the immune profile of these patients. Based on bioinformatics, a high PTGS2/CTLA4 ratio determines particularly dysfunctional NK cell states, along with the reduction of NK gene score associated with infiltration, cytotoxicity, and survival in HGSOC. Additionally, selective COX-2 blockade improves NK cell cytotoxicity against HGSOC cell lines independently of the CTLA-4 signaling. In fact, ipilimumab effect was almost absent in those cases with high COX2 activity. Interestingly, previous treatment with celecoxib on NK cells mainly explains the difference in cytotoxicity (p= 0.0253, two-way ANOVA test with Tukey’s multiple comparisons). Conclusions: This work represents the first evidence of the influence of COX-2 on NK cell function as a crucial factor for the efficacy of anti-CTLA-4 therapy in HGSOC patients who exhibited high tumoral levels of CTLA-4. HGSOC patients present dysfunctional circulating NK cells preferentially due to the strong CO-2 expression. Last, we emphasize the modulation of chronic inflammation to direct an immune pattern with greater antitumor capacity in HGSOC. (Funding: Fondecyt 1201083 and FONDAP 152220002 [MAC], CONICYT/Doctorado Nacional 2019-Folio 21190421 [FGV], CONICYT FONDAP 15130011 [MAC, IW]). Citation Format: Fernan Gomez-Valenzuela, Ignacio Wichmann, Felipe Suárez, Sumie Kato, Enrique Ossandón, Marcela Hermoso, Elmer Fernández, Mauricio A. Cuello. Cyclooxygenase-2 blockade is crucial to restore natural killer cell activity before anti-CTLA-4 therapy against high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 182.

Abstract 4059: Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment

Abstract Background: Interleukin-2 is the first approved immune-oncology (IO) drug to treat multiple metastatic cancers with durable and curative antitumor effects. Yet, its application is restricted due to severe toxicity, short half-life, and a small fraction of response rate. To reduce toxicity and improve efficacy, we developed Vitokine-2, a tumor infiltrating lymphocyte (TIL)-targeting, tumor activable, and precision engineered IL-2 that remains inert systemically but activated in the tumor microenvironment (TME). Methods: Through protein and structure-guided precision engineering, the biological activity of IL-2 was optimized, concealed, guided to exhausted TILs, and activated at TME. The biological activity was determined by measuring proliferation (Ki67) of CD8+ T and NK cells using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor efficacy were evaluated in mice, encompassing healthy and multiple syngeneic tumor mouse models. Results: Vitokine-2 was inactive in stimulating CD8+T and NK cell proliferation in vitro. Yet, its biological activity was fully restored following the cleavage and release of the concealing domain. Vitokine-2 displayed a significantly prolonged in vivo pharmacokinetic profile due to half-life extension, concealing and reduction of target sink. Administration of Vitokine-2 in mice resulted in minimal or no detectable activation, proliferation, and expansion of cytotoxic CD8+T and NK cells in the systemic circulation. However, marked CD8+ T cell proliferation and expansion with enhanced expression of granzyme B, a cytotoxic functional biomarker, was observed within the TME. The effect was specific to cytotoxic lymphocytes but not regulatory T cells. Corroborating with the expansion and enhanced functionality of CD8+ T cells in TME, robust antitumor efficacy was observed with Vitokine-2 but not with the non-activable IL-2 counterpart. Furthermore, dose-dependent tumor killing efficacy and complete eradication of tumors were demonstrated in multiple syngeneic tumor models. Vitokine-2 was found to be safe and well tolerated with minimal interferon gamma release and body weight loss even at much higher doses. Conclusion: Vitokine-2 is a TIL-targeting, tumor activable and precision engineered IL-2. It demonstrated robust anti-tumor efficacy with minimal systemic toxicity. It is safe, well tolerated, and permits high dose administration, yielding a widened therapeutic window. Vitokine-2 presents a paradigm-shifting, potentially a first-in-class, highly safe and efficacious IO therapy with an immense potential to address both IO-responsive and unresponsive patient population. Citation Format: Sreerupa V. Challa, Yao-Te Hsieh, Karen Gelinas, Andrea Umana, Wei-chun Weng, Toska Berisha, Cameron Stephens, Yuesheng Li, Jing Xu. Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4059.

The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

Abstract P20: Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia

Abstract P20: Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia

Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Dynamic immune signatures of patients with advanced non-small-cell lung cancer for infection prediction after immunotherapy.

Pulmonary infections are a crucial health concern for patients with advanced non-small-cell lung cancer (NSCLC). Whether the clinical outcome of pulmonary infection is influenced by immunotherapy(IO) remains unclear. By evaluating immune signatures, this study investigated the post-immunotherapy risk of pulmonary infection in patients with lung cancer and identified circulating biomarkers that predict post-immunotherapy infection. Blood specimens were prospectively collected from patients with NSCLC before and after chemotherapy(C/T) and/or IO to explore dynamic changes in immune signatures. Real-world clinical data were extracted from medical records for outcome evaluation. Mass cytometry and ELISA were employed to analyze immune signatures and cytokine profiles to reveal potential correlations between immune profiles and the risk of infection. The retrospective cohort included 283 patients with advanced NSCLC. IO was associated with a lower risk of pneumonia (odds ratio=0.46, p=0.012). Patients receiving IO and remained pneumonia-free exhibited the most favorable survival outcomes compared with those who received C/T or developed pneumonia (p<0.001). The prospective cohort enrolled 30 patients. The proportion of circulating NK cells significantly increased after treatment in IO alone (p<0.001) and C/T+IO group (p<0.01). An increase in cell densities of circulating PD-1+CD8+(cytotoxic) T cells (p<0.01) and PD-1+CD4+ T cells (p<0.01) were observed in C/T alone group after treatment. In IO alone group, a decrease in cell densities of TIM-3+ and PD-1+ cytotoxic T cells (p<0.05), and PD-1+CD4+ T cells (p<0.01) were observed after treatment. In C/T alone and C/T+IO groups, cell densities of circulating PD-1+ cytotoxic T cells significantly increased in patients with pneumonia after treatment(p<0.05). However, in IO alone group, cell density of PD-1+ cytotoxic T cells significantly decreased in patients without pneumonia after treatment (p<0.05). TNF-α significantly increased after treatment with IO alone (p<0.05) but decreased after C/T alone (p<0.01). Our results indicate that the incorporation of immunotherapy into treatment regimens may potentially offer protective effects against pulmonary infection. Protective effects are associated with reduction of exhausted T-cells and augmentation of TNF-α and NK cells. Exhausted T cells, NK cells, and TNF-α may play crucial roles in immune responses against infections. These observations highlight the potential utility of certain circulating biomarkers, particularly exhausted T cells, for predicting post-treatment infections.

TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Changes in immune status of circulating NK cells in patients with latent tuberculosis infection.

The presence of patients with latent tuberculosis infection (LTBI) has fueled the tuberculosis pandemic. We aimed to investigate the immune status of NK cells in LTBI patients. Twenty-one LTBI patients, 25 active pulmonary tuberculosis (APTB) patients and 25 healthy controls (HCs) participated in our research. The markers of NK cells were detected by flow cytometry. The absolute number of circulating CD56bright and CD56dim NK cells in LTBI patients was higher than that of APTB patients, but the frequency of HLA-DR+ CD56bright NK cells was significantly lower than that of HCs and APTB patients. Also, LTBI patients with CD56bright NK cells had intracellular levels of granzyme B that were as significantly elevated as those with APTB patients, but the levels of granzyme A and perforin were reduced. Meanwhile, the frequencies of CXCR3+ NK cells, CXCR3+ CD56bright and CXCR3+ CD56dim NK cells were significantly lower in LTBI patients. Circulating CD56bright NK cells exerted a significant role in maintaining immune balance in LTBI patients. An elevated frequency of granzyme B+ CD56bright NK cells and a reduced frequency of perforin+ CD56bright NK cells were effective in differentiating LTBI patients from HCs.

Cyclooxygenase-2 Blockade Is Crucial to Restore Natural Killer Cell Activity before Anti-CTLA-4 Therapy against High-Grade Serous Ovarian Cancer.

Chronic inflammation influences the tumor immune microenvironment (TIME) in high-grade serous ovarian cancer (HGSOC). Specifically, cyclooxygenase-2 (COX-2) overexpression promotes cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) expression. Notably, elevated COX-2 levels in the TIME have been associated with reduced response to anti-CTLA-4 immunotherapy. However, the precise impact of COX-2, encoded by PTGS2, on the immune profile remains unknown. To address this, we performed an integrated bioinformatics analysis using data from the HGSOC cohorts (TCGA-OV, n = 368; Australian cohort AOCS, n = 80; GSE26193, n = 62; and GSE30161, n = 45). Employing Gene Set Variation Analysis (GSVA), MIXTURE and Ecotyper cell deconvolution algorithms, we concluded that COX-2 was linked to immune cell ecosystems associated with shorter survival, cell dysfunction and lower NK cell effector cytotoxicity capacity. Next, we validated these results by characterizing circulating NK cells from HGSOC patients through flow cytometry and cytotoxic assays while undergoing COX-2 and CTLA-4 blockade. The blockade of COX-2 improved the cytotoxic capacity of NK cells against HGSOC cell lines. Our findings underscore the relevance of COX-2 in shaping the TIME and suggest its potential as a prognostic indicator and therapeutic target. Increased COX-2 expression may hamper the effectivity of immunotherapies that require NK cell effector function. These results provide a foundation for experimental validation and clinical trials investigating combined therapies targeting COX-2 and CTLA-4 in HGSOC.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

NK cells and the profile of inflammatory cytokines in the peripheral blood of patients with advanced carcinomas

BackgroundNatural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56brightCD16- (regulatory) and CD56dimCD16+ (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production. MethodsPeripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56brightCD16- and CD56dimCD16+ NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum. ResultsCancer patients had a lower frequency of the cytotoxic CD56dim CD16+ NK cells subset in comparison with healthy controls. Also, the regulatory CD56bright CD16- NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56brightCD16- NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56dimCD16+ was positively correlated with EGF and negatively correlated with MIP-1β. ConclusionsTaken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches.

Single-Cell RNA Sequencing Reveals the Interplay between Circulating CD4 + T Cells, B Cells and Cancer-Associated Monocytes in Classic Hodgkin Lymphoma Treated with PD-1 Blockade

Classic Hodgkin lymphoma (cHL) is a largely MHC class I-negative tumor with recurrent 9p24.1/ PD-L1/ PD-L2 copy gains and the highest reported response rates to PD-1 blockade. In cHL, the efficacy of PD-1 blockade is closely associated with Hodgkin Reed-Sternberg (HRS) cell expression of MHC class II, highlighting the potential role of CD4 + T-cell effectors and additional non-MHC class I-mediated mechanisms of tumor cell killing. We utilized scRNA and scT-cell receptor (TCR) sequencing to characterize the peripheral immune response to PD-1 blockade and define non-CD8 + T-cell dependent mechanisms of immune evasion in cHL. Peripheral blood mononuclear cells were obtained from 20 patients with relapsed/refractory (R/R) cHL who were treated with PD-1 blockade on the CheckMate 205 clinical trial (at cycle 1 day 1 [C1D1] and cycle 4 day 1 [C4D1]), 11 patients with newly diagnosed (ND), previously untreated cHL and 13 healthy donors. We first analyzed circulating CD4 + T cells given their likely importance in the response to PD-1 blockade in cHL. We found that patients with R/R cHL had markedly lower baseline TCR diversity than healthy donors across the entire CD4 + naïve/central memory (CM) T-cell space. Additionally, patients with the most favorable responses to PD-1 blockade had significantly increased CD4 + naïve/CM TCR diversity at baseline (C1D1) and more abundant naïve/CM subsets on treatment (C4D1). These response-related differences likely reflect a continued capacity to mount CD4 + T-cell responses to neoantigens. We also identified several circulating CD4 + T-cell populations that were significantly more abundant in patients with cHL than in healthy donors, including follicular helper-like T cells, IFN-stimulated cytotoxic T cells and actively proliferating CTLA4 + LAG3 + T cells. Compared to healthy donors, all evaluated patients with cHL had significantly reduced numbers of classically defined circulating NK cells and an expanded population of IFN-stimulated NK cells with likely reduced cytotoxic potential. In addition to marked deregulation of the NK cell compartment, all patients with cHL had decreased numbers of circulating B cells at all stages of differentiation. We used the scRNA sequencing data to reconstruct individual B-cell receptor (BCR) sequences with the TRUST4 algorithm and found that the numbers of total and unique BCRs, as well as chao1 diversity, were significantly decreased in patients with ND cHL in comparison to healthy donors. Additionally, patients with the most favorable responses to PD-1 blockade had significantly higher baseline BCR diversity and circulating B-cell numbers. Altogether, these data underscore the likely importance of B cell-mediated immune responses in cHL, either directly or via Fc-binding of innate effectors. Finally, the most abundant circulating CD3 - population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumor-promoting cytokines/chemokines, as well as PD-L1 and SIRPa. These inflamed IL1β + monocytes were virtually absent from the blood of healthy donors. Using RNAscope, we also detected macrophages with similar features in the intact cHL tumor microenvironment, in the immediate proximity of malignant HRS cells. To assess the broader significance of the inflamed IL1β + monocytes, we interrogated a single-cell compendium of monocytes and macrophages from multiple solid tumors and identified a population of monocytes/ macrophages with a similar transcriptional signature. These results suggest that similarly programmed inflammatory monocytes/macrophages are detectable in multiple cancers. We next assessed the clinical significance of circulating inflamed IL1β + monocytes in patients with R/R cHL who were treated with PD-1 blockade. Patients who did not respond to PD-1 blockade expressed significantly higher levels of the inflamed IL1β + monocyte transcriptional signature which led to the development of a predictive assay. We then identified a comparable circulating monocyte population with the same transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor, renal cell carcinoma, which underscored the broad significance of these findings. Taken together, our findings highlight the likely complementary role of CD4 + T cells, B cells and cancer-associated monocytes in the response to PD-1 blockade in cHL.