NK Cell Activity Research Articles

Updated results of a phase 1/2 study of AU-007, a monoclonal antibody (mAb) that binds to IL-2 and inhibits CD25 binding, in patients with advanced solid tumors.

2527 Background: AU-007 is a computationally designed human mAb that binds interleukin-2 (IL-2) on its CD25 binding epitope. AU-007 bound IL-2 cannot bind trimeric (CD25, CD122, CD132) IL-2 receptors (IL-2R) on regulatory T cells (Tregs), vascular endothelium, or eosinophils, but IL-2’s binding to dimeric IL-2R (CD122, CD132) on effector T (T eff) and NK cells is unhindered. AU-007 thus redirects IL-2 towards T eff and NK cell activation, while diminishing Treg activation and vascular leak. AU-007 uniquely redirects IL-2 generated from T eff cell expansion, converting a Treg-mediated autoinhibitory loop into an immune stimulating loop. Methods: The study consists of three dose escalation arms followed by cohort expansion. Arm 1A evaluates escalating doses (0.5 – 12 mg/kg) of AU-007 (IV every 2 weeks [Q2W]). Arm 1B evaluates AU-007 Q2W + escalating doses (15K – 270K IU/kg) of one aldesleukin subcutaneous (SC) dose. Arm 1C evaluates AU-007 + escalating doses of SC aldesleukin, both Q2W. Nineteen solid tumor types are allowed in escalation. Cohort expansion Arm 2B evaluates 9 mg/kg AU-007 + one 135K IU/kg aldesleukin dose. Results: Fifty-three patients were enrolled as of 23 January 2024: 15 in Arm 1A, 12 in Arm 1B, 25 in Arm 1C, and 1 in cohort expansion. AU-007 (+/- aldesleukin) was well-tolerated, with no dose limiting toxicities through all Arm 1A and 1B cohorts and the 3rd cohort (of 4) in 1C. Enrollment is ongoing in the final Arm 1C cohort and Arm 2B expansion. All drug related adverse events (AE) were Grade 1 or 2 except for 4 patients with transient lymphopenia (Grade 3 and 4) and 1 patient with Grade 3 anemia. The most common drug-related AEs were pyrexia (18%), fatigue (16%), nausea (14%), lymphopenia (8%), and chills (6%). Two patients had transient Grade 2 drug-related serious AEs: pyrexia Arm 1B and cytokine release syndrome Arm 1C; both patients continued therapy. A confirmed partial response (32% decrease) occurred in a nasopharyngeal carcinoma patient in Arm 1C who progressed on anti-PD-1 therapy. Tumor shrinkage occurred in patients with non-small cell lung, bladder, head and neck, colorectal (CRC), and renal cancer. A melanoma patient (Arm 1B) who progressed on anti-CTLA-4 + anti-PD-1 therapy had a 48% decrease in target tumors; a small brain metastasis was found and irradiated at week 16 and the patient remains on treatment. A patient with microsatellite stable CRC had a 26% tumor shrinkage after the first cycle (Arm 1C). Serum Tregs and eosinophils decreased in patients while NK and CD8 cell counts trended upwards. The CD8:Treg ratio trends upward in all cohorts. Conclusions: The mild toxicity profile and promising early efficacy observed in dose escalation across multiple tumor types in heavily pretreated patients, along with initial pharmacodynamic data, warrant continued evaluation of AU-007 + low dose SC aldesleukin in the Phase 2 expansion cohorts of the study. Clinical trial information: NCT05267626 .

Phase I dose escalation trial of the first-in-class TNFR2 agonist monoclonal antibody, HFB200301, in monotherapy and in combination with tislelizumab, an anti-PD-1 monoclonal antibody, in adult patients with advanced solid tumors.

2526 Background: Tumor necrosis factor receptor-2 (TNFR2) is expressed on effector CD8+, CD4+, and regulatory T (Treg) cells, natural killer (NK) cells, and myeloid cells. Activation of TNFR2 is anticipated to yield effective anti-tumor immunity by stimulating T-cell and NK-cell activation and proliferation in the tumor microenvironment. HFB200301 is a first-in-class anti-TNFR2 agonistic monoclonal antibody that triggers both innate and adaptive immune stimulation by binding to a specific epitope on TNFR2. We report initial results of an ongoing Phase I dose escalation, multicenter study (NCT05238883) of HFB200301 in monotherapy and in combination with tislelizumab (TIS) in patients (pts) with advanced refractory solid tumors. Methods: A Phase I trial designed to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD), and initial efficacy of HFB200301 in monotherapy and in combination with TIS. Eligible pts, ≥18 years with an ECOG PS 0-1 and advanced solid tumors, will be enrolled in dose escalation at 5 different dose cohorts in monotherapy (Q4W), 3 different dose cohorts in combination with TIS (Q4W), or in dose expansion following determination of safety. Radiographic response is assessed every 8 weeks. Results: A total of 39 pts have been enrolled, 27 pts in monotherapy and 12 pts in combination. All pts received prior systemic therapy (median 2, range 1-4); 23 pts (59%) had prior checkpoint inhibitor treatment. No dose limiting toxicities were observed in either the monotherapy or combination arms. Treatment-related adverse events (TRAEs) occurred in 44% of monotherapy and in 50% of combination pts, with pruritis (11%), tremor (7%), fatigue (7%), asthenia (7%) and nausea (7%) being the most common TRAEs. No ≥ Grade 3 TRAEs were reported, and there were no drug discontinuations due to TRAEs. Preliminary HFB200301 PK analysis demonstrated non-linear clearance of HFB200301, with dose-proportional maximum concentration [Cmax], achieving sufficient exposures for peripheral target engagement in all tested doses. Emerging PD data demonstrates evidence of immune activation and expansion of CD8+ T cells, but not Tregs, in the tumor microenvironment and peripheral blood. Single cell RNAseq analysis of peripheral blood mononuclear cells demonstrated PD modulation in TNFR2 positive immune cell types post-HFB200301 treatment. Early response evaluation reveals ongoing durable clinical responses (> 6 months) in a PD-L1+ pleural mesothelioma pt and an EBV+ gastric cancer pt from combination cohorts. Conclusions: HFB200301 demonstrates a favorable safety profile, dose-dependent PK, with PD and preliminary clinical activity in monotherapy and in combination with TIS in pts with heavily pre-treated refractory solid tumors. Clinical trial information: NCT05238883 .

Intravenous infusion (IV) or intracavitary perfusion (IP) of T3011, an oncolytic HSV expressing IL-12 and PD-1 antibody: Analyses of two phase 1 studies.

e14581 Background: T3011 is a recombinant HSV-1 oncolytic virus expressing both IL-12 and anti-human PD-1 antibody. Upon delivery, locally produced IL-12 and PD-1 induces IFN-γ production, enhances the oncolytic activity of NK cells and cytotoxic T lymphocytes, promotes anti-angiogenesis, and inhibits tumor growth. We present the results of T3011 monotherapy administered via IV in advanced solid tumors or IP for malignant pleural effusion/ascites. Methods: In a phase 1, dose-escalation and expansion study using a 3+3 design with 5 cohorts (1×106, 1×107, 1×108, 5×108, 1×109 PFU), T3011 was administered intravenously on Days 1, 4, 8, 11,15,18 of a 28-day cycle in pts with advanced solid tumors. The primary objective was to evaluate the safety and tolerability of IV T3011, characterize dose-limiting toxicities (DLTs), MTD or RP2D, pharmacokinetic and pharmacodynamic profiles. In another pilot study to evaluate the safety and efficacy of T3011 in pts with malignant pleural effusion or ascites, T3011 was infused into the pleural or peritoneal cavity through an indwelling catheter on Days 1, 4, 8, 11 with 2 cohorts (5×108, 2×109 PFU). The response of malignant pleural effusion or ascites was assessed by WHO criteria. Results: As of 15 Dec 2023, 18 pts were enrolled and treated with IV T3011,4 pts received IV T3011 therapy at the highest dose level, 1×109 PFU. T3011 was well tolerated without ≥ Grade 3 TRAEs or DLTs. MTD was not reached. The most common TRAEs (≥ 10%) were nausea and infusion-related reactions, diarrhea, etc. Among 14 evaluable pts with advanced solid tumors, 3 pts had confirmed SD per RECIST1.1 and tumor burden reduced significantly. All the pts had detectable T3011 DNA in blood within 0.5-2 hours after administration, which was cleared subsequently within 3-7 days. As of 5 Jan 2024, 6 pts (pleural effusion 3, ascites 3) were dosed with IP T3011. 2 pts received 5×108 PFU and 4 pts received 2×109 PFU. Most TEAEs were grade 1 or 2. Only 1 pt developed grade 3 leukopenia, which resolved with G-CSF. No SAE occurred. 3 pts with pleural effusion showed 1 CR, 1 PR and 1 SD. 3 pts with malignant ascites secondary to advanced colorectal cancer, 1 was evaluable with PR. Conclusions: IV T3011 monotherapy has a very manageable safety profile and some encouraging activity in advanced solid tumors. IP T3011 monotherapy is also well tolerated and demonstrates promising activity against malignant pleural effusion and ascites. Clinical trial information: NCT04780217 .

Preliminary results from a phase I expansion study of ZG005, a bispecific antibody targeting TIGIT and PD-1, as monotherapy in patients with advanced solid tumors.

2611 Background: ZG005, a PD-1 and TIGIT dual-specific antibody, is a promising immunotherapy for tumors as blocking these two pathways could synergistically activate T cells and enhance the anti-tumor activity of NK cells. Partial results from the dose-escalation stage of this first-in-human (FIH) study were revealed at ASCO 2023, and here we present the preliminary results from the dose-expansion stage. Methods: After a dose-escalation stage, patients (subjects) with solid advanced tumors were enrolled into this dose-expansion stage. Subjects in each cohort were randomized 1:1 to receive ZG005 treatment of either RP2D from the dose-escalation stage (10 mg/kg Q3W or 20 mg/kg Q3W) by intravenous infusion. Efficacy was assessed primarily according to RECIST v1.1. Results: The dose-escalation stage was completed with 32 subjects enrolled and the dose-expansion stage is currently ongoing. As of December 25, 2023, a total of 68 subjects were treated with ZG005 as monotherapy, including 33 males (median age 60) and 35 females (median age 58), wherein 33 had received PD-1/PD-L1 inhibitors before enrollment. Three subjects with either pancreatic neuroendocrine cancer, intrahepatic cholangiocarcinoma, or cervical cancer were treated for more than 20 cycles. The subject with cervical cancer maintained PR status for more than 35 weeks. A total of 1 CR, 6 PR, and 28 SD cases were reported from the 57 efficacy evaluable subjects, among them 1 CR, 5 PRs, and 8 SDs from the 17 subjects with cervical cancer. A total of 43 TRAEs (43/68, 63.2%) were reported, of which 6 were ≥ grade 3, including 2 liver function abnormalities, 1 elevated γ-glutamyl- transferase, 1 unknown death, 1 hypertriglyceridemia and lipase elevation, and 1 elevation in both aspartate aminotransferase and blood bilirubin. SAEs occurred in 19 subjects, 3 events were related to ZG005, including 2 liver function abnormalities (1 was a DLT event), and 1 unknown death. The death event occurred in one female, who received only one dose of ZG005, and the cause of her death was most likely due to poor underlying condition. The Cmax and AUC increased approximately in dose proportion. Conclusions: ZG005 has demonstrated a tolerable safety profile and encouraging anti-tumor activity during the FIH study. Expansion cohorts in specific advanced solid tumors are underway to warrant further development. Clinical trial information: CTR20220021.

Autologous, oncolytic virus-activated killer (VAK) cell therapy to treat malignant pleural and peritoneal effusions.

e20549 Background: OH2 is an attenuated oncolytic Herpes Simplex-2 virus expressing GM-CSF. OH2 replicates only in cancer cells, causing their lysis, which reverses the immunosuppressive tumor microenvironment, leading to tumor regression. OH2 monotherapy is safe and efficacious in phase I-III trials of 300+ patients across several cancer indications. We previously showed that OH2 treated briefly with UV light (UV-OH2) is unable to form plaques yet still able to infect cancer cells and to potently activate human PBMCs, leading to increased antitumor activity in vitro and in animal models. Methods: Translating these findings in humans, 12 stage IV lung cancer patients with malignant pleural or peritoneal effusions were enrolled in an Investigator-Initiated Trial (NCT05565014). Lymphocytes from each patient’s effusions were isolated, incubated in vitro with UV-OH2 for 36-48h, and then re-infused into the patient's pleural or peritoneal cavity. Each patient received 2 cycles of 3 or up to 6 infusions of this Virus Activated Killer (VAK) Autologous Cell Therapy. Results: Effusion volume (EV) measurement and local and whole-body tumor assessments were performed at the beginning and 28 days post VAK treatment. On D28, EV remained stable in 3 patients (SD), whereas 4 of 12 patients recorded a reduction of EV (PR). Notably, subject S004’s PR was maintained for 210 days post-treatment. Evaluation of target lesions per RECIST revealed 4 subjects with PR and 7 with an SD in target lesions. There was no toxicity or treatment-related adverse events. The median patient OS was 384 days (12.8 months), which is up to 4 times the average published OS for stage IV lung cancer with malignant pleural effusions. RNA-Seq analysis indicated increased expression of many T and NK cell effectors and NK cell activating receptors. Importantly, average autologous CD8+ T lymphocytes rose from 53% to 74% in the effusions post VAK-treatment, thereby confirming activation of the patient’s immune system by UV-OH2 (preliminary clinical PoC). Conclusions: In sum, our VAK therapy offers a cost-effective, extremely safe, and patient-friendly autologous treatment that dramatically increases overall survival in patients with essentially no options. Larger scale studies are in the works. Clinical trial information: NCT05565014 .

Tregs, gMDSCs, and levels of soluble MICA as prognostic biomarkers for combined NEO-201 and pembrolizumab.

2530 Background: The humanized IgG1 monoclonal antibody NEO-201 binds to Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, and mediates killing of cancer cells, neutrophils, regulatory T cells (Tregs) and granulocytic myeloid-derived suppressor cells (gMDSCs) via ADCC and CDC. Resistance to PD-1/PD-L1 blockade may be due to the accumulation of Tregs and gMDSCs in the tumor microenvironment (TME). NEO-201 was proven to bind and reduce the amount of circulating Tregs in cancer patients. This supports the rationale of the ongoing phase II clinical trial (NCT03476681) evaluating the activity of NEO-201 with pembrolizumab in adults with solid tumors resistant to prior checkpoint inhibitors. Furthermore, elevated serum levels of soluble MICA (sMICA) have been correlated with decreased NK cell activity. Methods: Cancer patients were treated each cycle with 3 doses of NEO-201 1.5mg/kg every 2 weeks and pembrolizumab 400mg IV every 6 weeks and imaged for response every 2 cycles. PBMCs and serum from cancer patients pre- and at multiple time points post-treatment were used to evaluate the percentage of circulating gMDSCs and Tregs (flow cytometry) and sMICA levels (ELISA). Results: NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) but not effector Tregs (eTregs: CD3+/CD4+/CD45RA-/Foxp3high cells). NEO-201 also binds to gMDSCs, defined as HLA-DRneg/CD33+/CD15+/ CD14neg/CD66b+ cells. In patients with durable SD, a patient with HNSCC (SD >11 months) showed >50% reduction of nTregs and >90% of gMDSCs at C3D1 (beginning of cycle 3) compared to baseline. One patient with cervical cancer (SD >8 months) showed 40% reduction of nTregs at C3D1 compared to baseline, while gMDSCs trended down to baseline levels at C3D1 after initial increase. Conversely, we observed a general uptrend of nTregs and gMDSCs in patients with PD after treatment. Median serum levels of sMICA pre-treatment were 33-fold higher in patients with PD compared to patients with SD. Levels of sMICA remained elevated in patients with PD and low in patients with SD at all time points post-treatment. Conclusions: Depletion of circulating Tregs and gMDSCs may prevent their accumulation in the TME and enhance the efficacy of pembrolizumab in subjects with tumors resistant to checkpoint inhibitors. The decrease in circulating nTregs and gMDSCs after treatment with NEO-201 and pembrolizumab was associated with durable SD. High levels of sMICA can impair ADCC mediated by NEO-201, resulting in poor clinical response. Low levels of sMICA in patients with SD, together with the reduction of both circulating nTregs and gMDSCs, could be favorable prognostic markers for clinical benefit following treatment with NEO-201 and pembrolizumab. Ongoing enrollment in this clinical trial will validate these findings in larger cohorts. Clinical trial information: NCT03476681 .

Study on the material basis and immunological enhancement activity of dangdi oral liquid

Studies have shown that a lot of traditional Chinese medicines could improve the immunity of the body. Dangdi oral liquid (DDO) was mainly composed of Angelica sinensis (Oliv.) Diels (Danggui), Rehmannia glutinosa Libosch. (Dihuang), Achyranthes bidentata Bl. (Niuxi), Glycyrrhiza uralensis Fisch. (Gancao). In this study, the rapid ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method was used to identify the potentially effective compounds of DDO. Then the immune activity of DDO was measured by lymphocyte proliferation, macrophage phagocytic function, NK cell activity, delayed type hypersensitivity reaction, hemolytic plaque number, sIgA content and immune organ index. The results showed that a total of 51 compounds were identified. In addition, DDO could significantly promote the lymphocyte proliferation, improve macrophage phagocytic ability, NK cell activity, hemolytic plaque number, sIgA content and immune organ index compared with control group, and the medium dose possessed the best efficacy (P＜0.05). These results indicated that DDO could enhance the immunity of mice.

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation.

The surface receptor CD8α is present on 20%-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α- (persistent CD8α-). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell-activating receptors.

Functional antibody responses targeting the Spike protein of SARS-CoV-2 Omicron XBB.1.5 in elderly nursing home residents following Wuhan-Hu-1-based mRNA booster vaccination

The immune effector mechanisms involved in protecting against severe COVID-19 infection in elderly nursing home residents following vaccination or natural infection are not well understood. Here, we measured SARS-CoV-2 Spike (S)-directed functional antibody responses, including neutralizing antibodies (NtAb) and antibody Fc-mediated NK cell activity (degranulation and IFNγ production), against the Wuhan-Hu-1, BA.4/5 (for NtAb), and Omicron XBB.1.5 variants in elderly nursing home residents (n = 39; median age, 91 years) before and following a third (pre- and post-3D) and a fourth (pre- and post-4D) mRNA COVID-19 vaccine dose. Both 3D and 4D boosted NtAb levels against both (sub)variants. Likewise, 3D and 4D increased the ability of sera to trigger both LAMP1- and IFNγ-producing NK cells, in particular against XBB.1.5. In contrast to NtAb titres, the frequencies of LAMP1- and IFNγ-producing NK cells activated by antibodies binding to Wuhan-Hu-1 and Omicron XBB.1.5 S were comparable at all testing times. Stronger functional antibody responses were observed in vaccine-experienced participants compared to vaccine-naïve at some testing times. These findings can contribute to identifying a reliable correlate of protection in elderly nursing home residents against severe COVID-19 and inform future vaccine strategies in this population group.

Immune micro-environment analysis and drug screening for ovarian endometriosis.

Patients of ovary endometriosis have an abnormal immune micro-environment, leading to endometrial tissue that from retrograde menstruation evade immune surveillance and subsequently develop into ectopic lesions. This study aims to elucidate the crucial immune cells and molecular pathways that are associated with an aberrant immune micro-environment of endometriosis. In this study, we identified differentially expressed genes between ovarian ectopic endometrial tissue (OVE) and eutopic endometrial tissue from patients with endometriosis (PE) and non-endometriosis patients (CON) by analyzing the mRNA sequencing data. Additionally, we used WGCNA(Weighted Gene Co-expression Network Analysis) to screen for key genes related to immune cell infiltration and compared the sub-types of infiltrating immune cells using CIBERSORT(cell-type identification by estimating relative subsets of RNA transcript). Subsequently, we conducted a single-cell analysis on the identified key genes. Furthermore, we analyzed potential drugs suitable for ovarian endometriosis treatment using pRRophertic. Seven key genes associated with immune cell infiltration were screened out. The expression of these genes in OVE was significantly lower than that in PE and CON. These key genes were mainly enriched in the NK cell-mediated cytotoxicity pathway, especially for CD16 + CD56dim NK. Moreover, NK cells infiltration in ovarian endometriosis was significantly reduced compared with PE and CON, while M2 macrophage shown the opposite. Results of the single-cell analysis showed that the expression of the seven key genes in NK cells and monocyte-macrophages in OVE was significantly lower than that in PE or CON. Additionally, we identified potential drugs suitable for ovarian endometriosis treatment. The decreased infiltration of NK cells and increased infiltration of M2 macrophages contribute to the evasion of immune surveillance against endometrial tissue, promoting the progression of OVE. Therefore, potential strategies for the treatment of OVE include increasing NK cell activation and decreasing M2 macrophage polarization.

Lack of TGFβ signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade.

Transforming growth factor beta (TGFβ) is well-recognized as an immunosuppressive player in the tumor microenvironment but also has a significant impact on cancer cell phenotypes. Loss of TGFβ signaling impairs DNA repair competency, which is described by a transcriptomic score, βAlt. Cancers with high βAlt have more genomic damage and are more responsive to genotoxic therapy. The growing appreciation that cancer DNA repair deficits are important determinants of immune response prompted us to investigate the association of βAlt with response to immune checkpoint blockade (ICB). We predicted that high βAlt tumors would be infiltrated with lymphocytes because of DNA damage burden and hence responsive to ICB. We analyzed public transcriptomic data from clinical trials and preclinical models using transcriptomic signatures of TGFβ targets, DNA repair genes, tumor educated immune cells and interferon. A high βAlt, immune poor mammary tumor derived transplant model resistant to programmed death ligand 1 (PD-L1) antibodies was studied using multispectral flow cytometry to interrogate the immune system. Metastatic bladder patients in IMvigor 210 who responded to ICB had significantly increased βAlt scores and experienced significantly longer overall survival compared to those with low βAlt scores (hazard ratio 0.62, P=0.011) . Unexpectedly, 75% of high βAlt cancers were immune poor as defined by low expression of tumor educated immune cell and interferon signatures. The association of high βAlt with immune poor cancer was also evident in TCGA and preclinical cancer models. We used a high βAlt, immune poor cancer to test therapeutic strategies to overcome its inherent anti-PD-L1 resistance. Combination treatment with radiation and TGFβ inhibition were necessary for lymphocytic infiltration and activated NK cells were required for ICB response. Bioinformatic analysis identified high βAlt, immune poor B16 and CT26 preclinical models and paired biopsies of cancer patients that also demonstrated NK cell activation upon response to ICB. Our studies support βAlt as a biomarker that predicts response to ICB albeit in immune poor cancers, which has implications for the development of therapeutic strategies to increase the number of cancer patients who will benefit from immunotherapy.

NK-cell receptor modulation in viral infections.

Natural killer (NK) cells play a crucial role in controlling viral infections. The ability to kill infected cells without prior immunization, yet being tolerant to self, healthy cells, depends on the balance of germ-line encoded surface receptors. NK-cell receptors are divided into either activating, leading to activation of NK cell and its cytotoxic and pro-inflammatory activity, or inhibitory, providing tolerance for a target cell. The signals from inhibitory receptors dominate and NK-cell activation requires stimulation of activating receptors. In viral infections, NK-cell interaction with infected cells can result in activation, memory-like NK-cell differentiation, or NK-cell exhaustion, which constitutes one of the viral immune evasion mechanisms. All of these states are associated with the modulation of NK-cell receptor expression. In this review, we summarize the current knowledge of NK-cell receptors and their role in viral infection control, as well as the alterations of their expression observed in acute or chronic infections. We present recently discovered SARS-CoV-2-mediated modulation of NK-cell receptor expression and compare them with other human viral infections. Finally, since modulation of NK-cell receptor activation gives a promising addition to currently used antiviral therapies, we briefly discuss the clinical significance and future perspective of the application of agonists or antagonists of activating and inhibitory receptors, respectively. In sum, our review shows that although much is known about NK-cell receptor biology, a deeper understanding of NK-cell receptors role in viral infections is still needed.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Lack of the IFN-γ signal leads to lethal Orientia tsutsugamushi infection in mice with skin eschar lesions.

Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/- and Stat1-/- mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, which resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-γ, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of innate IFN-γ, contributing to the initial Ot control in the draining lymph nodes. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-γ than IFN-β. Comparative in vivo studies with Karp- and Gilliam-infection revealed a crucial role of IFN-γ signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus.

Advances in Hodgkin Lymphoma Treatment: From Molecular Biology to Clinical Practice.

Classical Hodgkin Lymphoma (cHL) is a highly curable disease, but around 20% of patients experience progression or relapse after standard frontline chemotherapy regimens. Salvage regimens followed by autologous stem cell transplants represent the historical treatment approach for these cases. In the last decade, with the increasing understanding of cHL biology and tumor microenvironment role in disease course, novel molecules have been introduced in clinical practice, improving outcomes in the relapsed/refractory setting. The anti-CD30 antibody-drug conjugated brentuximab vedotin and PD-1/PD-L1 checkpoint inhibitors represent nowadays curative options for chemorefractory patients, and randomized trials recently demonstrated their efficacy in frontline immune-chemo-combined modalities. Several drugs able to modulate the patients' T-lymphocytes and NK cell activity are under development, as well as many anti-CD30 chimeric antigen receptor T-cell products. Multiple tumor aberrant epigenetic mechanisms are being investigated as targets for antineoplastic compounds such as histone deacetylase inhibitors and hypomethylating agents. Moreover, JAK2 inhibition combined with anti-PD1 blockade revealed a potential complementary therapeutic pathway in cHL. In this review, we will summarize recent findings on cHL biology and novel treatment options clinically available, as well as promising future perspectives in the field.

Fate control engagement augments NK cell responses in LV/hu-IL-12 transduced sarcoma

IntroductionNK cells are an untapped resource for cancer therapy. Sarcomas transduced with lentiviruses to express human IL-12 are only cleared in mice bearing mature human NK cells. However, systemic inflammation limits IL-12 utilization. Fate control a.k.a. “suicide mechanisms” regulate unchecked systemic inflammation caused by cellular immunotherapies. Despite increasing utilization, there remains limited data on immune consequences or tumor-directed effects of fate control. ObjectivesWe sought to engage the mutant thymidylate kinase (mTMPK) metabolic fate control system to regulate systemic inflammation and assess the impact on NK cell effector functions. MethodsPrimary human sarcoma short-passage samples and cell lines were transduced with LV/hu-IL-12_mTMPK engineering expression of IL-12 and an AZT-associated fate control enzyme. We assessed transduced sarcoma responses to AZT engagement and subsequent modulation of NK cell functions as measured by inflammatory cytokine production and cytotoxicity. ResultsAZT administration to transduced (LV/hu-IL-12_mTMPK) short-passage primary human sarcomas and human Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines, abrogated the robust expression of human IL-12. Fate control activation elicited a specific dose-dependent cytotoxic effect measured by metabolic activity (WST-1) and cell death (Incucyte). NK effector functions of IFN-γ and cytotoxic granule release were significantly augmented despite IL-12 abrogation. This correlated with preferentially induced expression of NK cell activation ligands. ConclusionsmTMPK fate control engagement terminates transduced sarcoma IL-12 production and triggers cell death, but also augments an NK cell-mediated response coinciding with metabolic stress activating surface ligand induction. Fate control engagement could offer a novel immune activation method for NK cell-mediated cancer clearance.

Development of a quantification method for arginase inhibitors by LC-MS/MS with benzoyl chloride derivatization

Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 μm, 30 × 3 mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1→176.1, 439.4→181.2, 334.9→150.0 and 339.9→150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00–10,000 ng/mL with QC values of 5, 50 and 500 ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.

HDAC Inhibition Increases CXCL12 Secretion to Recruit Natural Killer Cells in Peripheral T-cell Lymphoma.

Peripheral T-cell lymphoma (PTCL) is a heterogeneous and aggressive disease with a poor prognosis. Histone deacetylase (HDAC) inhibitors have shown inhibitory effects on PTCL. A better understanding of the therapeutic mechanism underlying the effects of HDAC inhibitors could help improve treatment strategies. Herein, we found that high expression of HDAC3 is associated with poor prognosis in PTCL. HDAC3 inhibition suppressed lymphoma growth in immunocompetent mice but not in immunodeficient mice. HDAC3 deletion delayed the progression of lymphoma, reduced the lymphoma burden in the thymus, spleen, and lymph nodes, and prolonged the survival of mice bearing N-methyl-N-nitrosourea-induced lymphoma. Furthermore, inhibiting HDAC3 promoted the infiltration and enhanced the function of natural killer (NK) cells. Mechanistically, HDAC3 mediated ATF3 deacetylation, enhancing its transcriptional inhibitory activity. Targeting HDAC3 enhanced CXCL12 secretion through an ATF3-dependent pathway to stimulate NK-cell recruitment and activation. Finally, HDAC3 suppression improved the response of PTCL to conventional chemotherapy. Collectively, this study provides insights into the mechanism by which HDAC3 regulates ATF3 activity and CXCL12 secretion, leading to immune infiltration and lymphoma suppression. Combining HDAC3 inhibitors with chemotherapy may be a promising strategy for treating PTCL. Significance: Targeting HDAC3 suppresses progression of T-cell lymphoma by activating ATF3 to induce secretion of CXCL12 and promote infiltration of NK cells, providing an immunostimulatory approach for treating T-cell lymphoma patients.

Abstract PO2-27-07: The Role of HCMV Infection in Facilitating Brain Metastasis through the Creation of a Suppressive Tumor Microenvironment in Breast Cancer Patients

Abstract Human Cytomegalovirus (HCMV) infection has emerged as a noteworthy factor in breast cancer patients, particularly those experiencing brain metastasis. Despite being prevalent and often asymptomatic, HCMV's contribution to tumor progression remains enigmatic. Notably, a high proportion of breast cancer patients with brain metastasis exhibit HCMV positivity in both primary tumors and metastatic lesions. This study aims to elucidate the influence of HCMV infection on brain metastasis by investigating its impact on the tumor microenvironment. Our findings reveal a distinctive immune suppressive tumor microenvironment in HCMV-positive breast cancer patients. This environment is characterized by impaired NK cell function and an elevated accumulation of TH2 cells. To comprehend the role of HCMV in promoting brain metastasis, we have developed a murine breast tumor model using MCMV, enabling us to mirror the clinical trajectory of tumor progression and metastasis. The integration of a GFP reporter gene into the MCMV genome facilitates the tracking of infected cancer cells. Within this model, we observe a significant increase in brain metastasis coupled with reduced survival rates. Single-cell sequencing analysis uncovers diminished NK cell presence and activation, along with heightened TH2 cell accumulation within the tumor microenvironment—results that parallel the clinical observations in breast cancer patients. GSEA analysis underscores the secretion of inhibitory cytokines by MCMV-infected cancer cells, contrasting with their non-infected counterparts. Collectively, our study reveals that CMV infection contributes to brain metastasis in breast cancer by generating inhibitory cytokines. This hampers the recruitment and proliferation of immune cells within the tumor microenvironment and concurrently impairs the cytotoxic capabilities of NK cells. These insights shed light on the intricate interplay between viral infection and tumor progression, offering potential avenues for therapeutic exploration. Citation Format: Wenjuan Dong, Xin Wang, Kun Han, Akshjot Puri, Amna Irfan, Wei Qian, Liliana Guzman, Roberto Rosato, Jianting Sheng, Hong Zhao, Jenny Chang, Stephen Wong. The Role of HCMV Infection in Facilitating Brain Metastasis through the Creation of a Suppressive Tumor Microenvironment in Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-07.

IL-12p35 and IL-12p40 functionally synergize to active NK cells

Abstract IL-12 plays critical roles in regulating the immune response toward different effector pathways. The formation of functional IL-12 requires association of the IL-12p35 subunit with the IL-12p40 subunit. IL-12p40 can be released from these hematopoietic cells as a monomer. Previous studies have reported that IL-12p40 associates with p35 extracellularly to generate IL-12 like activities on T cells, but the biological significance on NK cells remains unclear. In this study, we show that only high concentrations of IL-12p35 could activate NK cells to secrete IFN-gamma and induce cell proliferation and IL-12p40 had no effect on NK cell activation. Interestingly, IL-12p35 and p40 functionally synergized to activate NK cells by inducing IFN-gamma secretion and cell proliferation in vitro. Surprisingly, pretreatment of NK cells with IL-12p35 or p40 didn’t affect IL-12 induced IFN-gamma secretion and cell proliferation in NK cells. Furthermore, a functional ELISA binding assay revealed that IL-12p35 binds to both IL-12R beta1 and IL-12R beta2 with a low affinity, while IL-12p40 only binds to IL-12R beta1 with a high affinity. Fluorescent conjugated human IL-12, IL-12p35 and IL-12p40 proteins all significantly bound to human NK cells and IL-12p35 and p40 didn’t significantly interfere with IL-12 binding to these cells. Collectively, the data we show herein indicates that IL-12 activity can come from the collaboration between the IL-12p35 and p40 subunits and impact NK cell activation.