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The role of different biological variables including biological sex, age, and sex hormones in Human immunodeficiency virus (HIV) cure approaches is not well understood. The γc-cytokine IL-15 is a clinically relevant cytokine that promotes immune activation and mediates HIV reactivation from latency. In this work, we examined the interplay that biological sex, age, and sex hormones 17β-estradiol, progesterone, and testosterone may have on the biological activity of IL-15. We found that IL-15-mediated CD4+ T cell activation was higher in female donors than in male donors. This difference was abrogated at high 17β-estradiol concentration. Additionally, there was a positive correlation between age and both IL-15-mediated CD8+ T cell activation and IFN-γ production. In a primary cell model of latency, biological sex, age, or sex hormones did not influence the ability of IL-15 to reactivate latent HIV. Finally, 17β-estradiol did not consistently affect reactivation of translation-competent reservoirs in CD4+ T cells from people living with HIV who are antiretroviral therapy (ART) suppressed. Our study has found that biological sex and age, but not sex hormones, may influence some of the biological activities of IL-15. Understanding how different biological variables may affect HIV cure therapies will help us evaluate current and future clinical trials aimed toward HIV cure in diverse populations.

An insufficient quantity of functional T cells is a likely factor limiting the clinical activity of T-cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T-cell dependent (TDB) antibodies. The activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in the upregulation of the IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to IL-15. XmAb24306 enhanced T-cell bispecific antibody-induced CD8+ and CD4+ T-cell proliferation and expansion. In vitro combination of XmAb24306 with cevostamab or anti-HER2/CD3 TDB resulted in significant enhancement of tumor cell killing, which was reversed when T-cell numbers were normalized, suggesting that T-cell expansion is the main mechanism of the observed benefit. Pretreatment of immunocompetent mice with a mouse-reactive surrogate of XmAb24306 (mIL-15-Fc) resulted in a significant increase of T cells in the blood, spleen, and tumors and converted transient anti-HER2/CD3 TDB responses to complete durable responses. In summary, our results support the hypothesis that the number of tumor-infiltrating T cells is rate limiting for the activity of solid tumor-targeting TDBs. Upregulation of CD122 by TDB treatment and the observed synergy with XmAb24306 and T-cell bispecific antibodies support clinical evaluation of this novel immunotherapy combination., (©2024 American Association for Cancer Research.)

Infectious, inflammatory and autoimmune conditions present differently in males and females. SARS-CoV-2 infection in naive males is associated with increased risk of death, whereas females are at increased risk of long COVID 1 , similar to observations in other infections 2 . Females respond more strongly to vaccines, and adverse reactions are more frequent 3 , like most autoimmune diseases 4 . Immunological sex differences stem from genetic, hormonal and behavioural factors 5 but their relative importance is only partially understood 6-8 . In individuals assigned female sex at birth and undergoing gender-affirming testosterone therapy (trans men), hormone concentrations change markedly but the immunological consequences are poorly understood. Here we performed longitudinal systems-level analyses in 23 trans men and found that testosterone modulates a cross-regulated axis between type-I interferon and tumour necrosis factor. This is mediated by functional attenuation of type-I interferon responses in both plasmacytoid dendritic cells and monocytes. Conversely, testosterone potentiates monocyte responses leading to increased tumour necrosis factor, interleukin-6 and interleukin-15 production and downstream activation of nuclear factor kappa B-regulated genes and potentiation of interferon-γ responses, primarily in natural killer cells. These findings in trans men are corroborated by sex-divergent responses in public datasets and illustrate the dynamic regulation of human immunity by sex hormones, with implications for the health of individuals undergoing hormone therapy and our understanding of sex-divergent immune responses in cisgender individuals., (© 2024. The Author(s).)

The formation of memory T cells is a fundamental feature of adaptative immunity, allowing the establishment of long-term protection against pathogens. Although emerging evidence suggests that metabolic reprogramming is crucial for memory T cell differentiation and survival, the underlying mechanisms that drive metabolic rewiring in memory T cells remain unclear. Here, we found that up-regulation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) instructs the metabolic reprogramming that occurs during the establishment of central memory CD8 + T cells. PPARβ/δ-regulated changes included suppression of aerobic glycolysis and enhancement of oxidative metabolism and fatty acid oxidation. Mechanistically, exposure to interleukin-15 and expression of T cell factor 1 facilitated activation of the PPARβ/δ pathway, counteracting apoptosis induced by antigen clearance and metabolic stress. Together, our findings indicate that PPARβ/δ is a master metabolic regulator orchestrating a metabolic switch that may be favorable for T cell longevity.

Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM., Competing Interests: Declaration of interests M.S., M. Daher, R.B., H.R., S.A., Y.L., N.U., E.L., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical. R.B., E.J.S., K.R., and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Affimed GmbH. K.R. participates on the Scientific Advisory Board for Avenge Bio, Virogin Biotech, Navan Technologies, Caribou Biosciences, Bit Bio Limited, Replay Holdings, oNKo Innate, and The Alliance for Cancer Gene Therapy ACGT. K.R. is the Scientific founder of Syena. E.J.S. has served on the Scientific Advisory Board for Adaptimmune, Axio, Celaid, FibroBiologics, Navan Technologies, New York Blood Center and Novartis. M Daher participates on the Scientific Advisory Board for Cellsbin. M.S., H.S., E.J.S., and K.R. have filed for a patent (20230074303); “Cell immunotherapy for the treatment of cancer.”, (Copyright © 2024 Elsevier Inc. All rights reserved.)

NK cells are cytotoxic innate immune cells involved in antitumor immunity, and they provide a treatment option for patients with acute myeloid leukemia (AML). In this issue of the JCI, Cubitt et al. investigated the role of CD8α, a coreceptor present on approximately 40% of human NK cells. IL-15 stimulation of CD8α- NK cells induced CD8α expression via the RUNX3 transcription factor, driving formation of a unique induced CD8α (iCD8α+) population. iCD8α+ NK cells displayed higher proliferation, metabolic activity, and antitumor cytotoxic function compared with preexisting CD8α+ and CD8α- subsets. Therefore, CD8α expression can be used to define a potential dynamic spectrum of NK cell expansion and function. Because these cells exhibit enhanced tumor control, they may be used to improve in NK cell therapies for patients with AML.

Natural killer cells (NK cells) are the front line of immune cells to combat pathogens and able to influence the subsequent adaptive immune responses. One of the factors contributing to pathogenesis in dengue hemorrhagic fever (DHF) disease is aberrant immune activation during early phase of infection. This study explored the profile of NK cells in dengue infected pediatric patients with different degrees of disease severity. DHF patients contained higher frequency of activated NK cells but lower ratio of CD56 dim :CD56 bright NK subsets. Activated NK cells exhibited alterations in several NK receptors. Interestingly, the frequencies of NKp30 expressing activated NK cells were more pronounced in dengue fever (DF) than in DHF pediatric patients. In vitro functional analysis indicated that degranulation of NK cells in responding to dengue infected dendritic cells (DCs) required cell-cell contact and type I IFNs. Meanwhile, Interferon gamma (IFN-γ) production initially required cell-cell contact and type I IFNs followed by Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-18 (IL-18) resulting in the amplification of IFN-γ producing NK cells over time. This study highlighted the complexity and the factors influencing NK cells responses to dengue virus. Degree of activation, phenotypes of activated cells and the crosstalk between NK cells and other immune cells, could modulate the outcome of NK cells function in the dengue disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56 bright or CD56 dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined in vitro conditions that could polarize human NK cells into an inhibitory subset. TGF-β1 has been shown to induce regulatory T cells in vitro and in vivo; we therefore investigated if TGF-β1 could also induce immunosuppressive NK-like cells. First, we found that TGF-β1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells in vitro in a TGF-β-dependent manner. Interestingly, TGF-β1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells in vitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-β1-rich environments., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Cytokines are co-administrated with vaccines or co-expressed in the vaccine virus genome to improve protective efficacy by stimulating immune responses. Using glycosylphosphatidylinositol (GPI) anchoring by attachment to the target cytokine, we constructed recombinant Marek's disease virus (MDV) vaccine strain 301B/1 (v301B/1-rtg-IL-15) that expresses chicken interleukin-15 (IL-15) as the membrane-bound form at the cell surface. We evaluated the vaccine efficacy of v301B/1-rtg-IL-15 given as a bivalent Marek's disease (MD) vaccine in combination with turkey herpesvirus (HVT) against a very virulent plus MDV strain 648A challenge. The efficacy was compared with that of conventional bivalent MD vaccine, as a mixture with HVT plus parental v301B/1 or v301B/1-IL-15, which expresses a natural form of IL-15. The membrane-bound IL-15 expression did not interfere with the virus growth of recombinant v301B/1-rtg-IL-15. However, the MD incidence in birds vaccinated with v301B/1-rtg-IL-15 was higher than that of birds given the conventional bivalent MD vaccine containing parental v301B/1 virus, although the v301B/1-rtg-IL-15 vaccinated group showed increased natural killer cell activation at day 5 postvaccination, the same day as challenge. Overall, the protection of v301B/1-rtg-IL-15 was not improved from that of v301B/1 against very virulent plus MDV challenge.

Idiopathic severe aplastic anemia (SAA) is a disease of bone marrow failure caused by T-cell-induced destruction of hematopoietic stem and progenitor cells (HSPCs), however the mechanism remains unclear. We performed single-cell RNA sequencing of PBMCs and BMMCs from SAA patients and healthy donors and identified a CD8 + T cell subset with a tissue residency phenotype (Trm) in bone marrow that exhibit high IFN-γ and FasL expression and have a higher ability to induce apoptosis in HSPCs in vitro through FasL expression. CD8 + Trm cells were induced by IL-15 presented by IL-15Rα on monocytes, especially CD16 + monocytes, which were increased in SAA patients. CD16 + monocytes contributed to IL-15-induced CD38 + CXCR6 + pre-Trm differentiation into CD8 + Trm cells, which can be inhibited by the CD38 inhibitor 78c. Our results demonstrate that IL-15-induced CD8 + Trm cells are pathogenic cells that mediate HSPC destruction in SAA patients and are therapeutic targets for future treatments., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Interleukin 15 (IL-15) has emerged as a crucial factor in the relationship between natural killer (NK) cells and immunotherapy for cancer. This review article aims to provide a comprehensive understanding of the role of IL-15 in NK cell-mediated immunotherapy. First, the key role of IL-15 signaling in NK cell immunity is discussed, highlighting its regulation of NK cell functions and antitumor properties. Furthermore, the use of IL-15 or its analogs in clinical trials as a therapeutic strategy for various cancers, including the genetic modification of NK cells to produce IL-15, has been explored. The potential of IL-15-based therapies, such as chimeric antigen receptor (CAR) T and NK cell infusion along with IL-15 in combination with checkpoint inhibitors and other treatments, has been examined. This review also addresses the challenges and advantages of incorporating IL-15 in cell-based immunotherapy. Additionally, unresolved questions regarding the detection and biological significance of the soluble IL-15/IL-15Rα complex, as well as the potential role of IL-15/IL-15Rα in human cancer and the immunological consequences of prolonged exposure to soluble IL-15 for NK cells, are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

The clinical applications of immunocytokines are severely restricted by dose-limiting toxicities. To address this challenge, here we propose a next-generation immunocytokine concept involving the design of LH05, a tumor-conditional anti-PD-L1/interleukin-15 (IL-15) prodrug. LH05 innovatively masks IL-15 with steric hindrance, mitigating the "cytokine sink" effect of IL-15 and reducing systemic toxicities associated with wild-type anti-PD-L1/IL-15. Moreover, upon specific proteolytic cleavage within the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. Mechanistically, the antitumor efficacy of LH05 depends on the increased infiltration of CD8 + T and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors. Additionally, the tumor-conditional anti-PD-L1/IL-15 can synergize with an oncolytic virus or checkpoint blockade in advanced and metastatic tumor models. Our findings provide a compelling proof of concept for the development of next-generation immunocytokines, contributing significantly to current knowledge and strategies of immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The autoimmunity-promoting cytokine, Interleukin-15 (IL-15), is often claimed to be a key pathogenic cytokine in alopecia areata (AA). Yet, rhIL-15 promotes human hair follicle (HF) growth ex vivo. We have asked whether the expression of IL-15 and its receptor (IL-15R) isoforms is altered in human AA and how IL-15 impacts on human HF immune privilege (HF-IP) in the presence/absence of interferon-γ (IFNγ), the well-documented key AA-pathogenic cytokine, as well as on hair regrowth after experimental AA induction in vivo. Quantitative immunohistomorphometry showed the number of perifollicular IL-15 + T cells in AA skin biopsies to be significantly increased compared to healthy control skin, while IL-15, IL-15Rα, and IL-15Rγ protein expression within the hair bulb were significantly down-regulated in AA HFs. In organ-cultured human scalp HFs, rhIL-15 significantly reduced hair bulb expression of MICA, the key "danger" signal in AA pathogenesis, and increased production of the HF-IP guardian, α-MSH. Crucially, ex vivo, rhIL-15 prevented IFNγ-induced HF-IP collapse, restored a collapsed HF-IP by IL-15Rα-dependent signaling (as documented by IL-15Rα-silencing), and protected AA-preventive immunoinhibitory iNKT10 cells from IFNγ-induced apoptosis. rhIL-15 even promoted hair regrowth after experimental AA induction in human scalp skin xenotransplants on SCID/beige mice in vivo. Our data introduce IL-15 as a novel, functionally important HF-IP guardian whose signaling is constitutively defective in scalp HFs of AA patients. Our data suggest that selective stimulation of intrafollicular IL-15Rα signaling could become a novel therapeutic approach in AA management, while blocking it pharmacologically may hinder both HF-IP restoration and hair re-growth and may thus make HFs more vulnerable to AA relapse., Competing Interests: Declaration of Competing Interest None declared. For the record, RP and JG are employees of, and JC and AG consult for a company that develops hair growth-modulatory agents (www.cutaneon.com)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities., Methods: Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo., Results: Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion., Conclusion: This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 van Hees, Morton, Remst, Wouters, Van den Eynde, Falkenburg and Heemskerk.)

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood 1-4 . Here we use systems immunology approaches, including longitudinal multimodal single-cell analysis (surface proteins, transcriptome and V(D)J sequences) to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean, 151 days after diagnosis) and 40 age- and sex-matched control individuals who had never had COVID-19. At the baseline and independent of time after COVID-19, recoverees had elevated T cell activation signatures and lower expression of innate immune genes including Toll-like receptors in monocytes. Male individuals who had recovered from COVID-19 had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared with healthy male individuals and female individuals who had recovered from COVID-19, in part because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated prevaccination frequencies of 'virtual memory'-like CD8 + T cells poised to produce more IFNγ after IL-15 stimulation. Moreover, the expression of the repressed innate immune genes in monocytes increased by day 1 to day 28 after vaccination in recoverees, therefore moving towards the prevaccination baseline of the healthy control individuals. By contrast, these genes decreased on day 1 and returned to the baseline by day 28 in the control individuals. Our study reveals sex-dimorphic effects of previous mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points that affect future immune responses in an antigen-agnostic manner., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Introduction: Hantaan virus (HTNV) can cause endothelium injury in hemorrhagic fever with renal syndrome (HFRS) patients. Bystander activation of CD8+ T cells by virus infection has been shown that was involved in host injury, but it is unclear during HTNV infection. This project aimed to study the effect of bystander-activated CD8+ T cell responses in HTNV infection., Methods: The in vitro infection model was established to imitate the injury of endothelium in HFRS patients. Flow cytometry was performed to detect the expression of markers of tetramer+ CD8+ T cells and human umbilical vein endothelial cells (HUVECs). The levels of interleukin-15 (IL-15) in serum and supermanant were detected using ELISA kit. The expression of MICA of HUVECs was respectively determined by flow cytometry and western blot. The cytotoxicity of CD8+ T cells was assessed through the cytotoxicity assay and antibody blocking assay., Results: EBV or CMV-specific CD8+ T cells were bystander activated after HTNV infection in HFRS patients. HTNV-infected HUVECs in vitro could produce high levels of IL-15, which was positively correlated with disease severity and the expression of NKG2D on bystander-activated CD8+ T cells. Moreover, the elevated IL-15 could induce activation of CD122 (IL-15Rβ)+NKG2D+ EBV/CMV-specific CD8+ T cells. The expression of IL-15Rα and ligand for NKG2D were upregulated on HTNV-infected HUVECs. Bystander-activated CD8+ T cells could exert cytotoxicity effects against HTNV-infected HUVECs, which could be enhanced by IL-15 stimulation and blocked by NKG2D antibody., Discussion: IL-15 induced bystander activation of CD8+ T cells through NKG2D, which may mediate endothelium injury during HTNV infection in HFRS patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Zhang, Liu, Tang, Zhang, Wang, Xue, Jia, Hu, Li, Zhuang, Jin, Zhang, Zhang and Ma.)

IL-15 induces the proliferation of memory CD8 + T cells as well as NK cells. The expression of CD5 inversely correlates with the IL-15 responsiveness of human memory CD8 + T cells. However, whether CD5 directly regulates IL-15-induced proliferation of human memory CD8 + T cells is unknown. In the current study, we demonstrate that human memory CD8 + T cells in advanced stages of differentiation respond to IL-15 better than human memory CD8 + T cells in stages of less differentiation. We also found that the expression level of CD5 is the best correlate for IL-15 hyporesponsiveness among human memory CD8 + T cells. Importantly, we found that IL-15-induced proliferation of human memory CD8 + T cells is significantly enhanced by blocking CD5 with Abs or knocking down CD5 expression using small interfering RNA, indicating that CD5 directly suppresses the IL-15-induced proliferation of human memory CD8 + T cells. We also found that CD5 inhibits activation of the mTOR pathway, which is required for IL-15-induced proliferation of human memory CD8 + T cells. Taken together, the results indicate that CD5 is not just a correlative marker for IL-15 hyporesponsiveness, but it also directly suppresses IL-15-induced proliferation of human memory CD8 + T cells by inhibiting mTOR pathways., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-γ) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. IMPORTANCE Several clinical trials targeting the HIV latent reservoir with LRAs have been completed. In spite of a lack of clinical benefit, they have been crucial to elucidate hurdles that "shock and kill" strategies have to overcome to promote an effective reduction of the latent reservoir to lead to a cure. These hurdles include low reactivation potential mediated by LRAs, the negative influence of some LRAs on the activity of natural killer and effector CD8 T cells, an increased resistance to apoptosis of latently infected cells, and an exhausted immune system due to chronic inflammation. To that end, finding therapeutic strategies that can overcome some of these challenges could improve the outcome of shock and kill strategies aimed at HIV eradication. Here, we show that the LRA HODHBt also improves IL-15-mediated NK cell effector and memory-like functions. As such, pharmacological enhancement of IL-15-mediated STAT activation can open new therapeutic avenues toward an HIV cure.

Inducing a full antitumor immune response in the tumor microenvironment (TME) is essential for successful cancer immunotherapy. Here, we report that an oncolytic adenovirus carrying mIL-15 (Ad-IL15) can effectively induce antitumor immune response and inhibit tumor growth in a mouse model of cancer. We found that Ad-IL15 facilitated the activation and infiltration of immune cells, including dendritic cells (DCs), T cells and natural killer (NK) cells, in the TME. Unexpectedly, we observed that Ad-IL15 also induced vascular normalization and tertiary lymphoid structure formation in the TME. Moreover, we demonstrated these Ad-IL15-induced changes in the TME were depended on the Ad-IL15-induced activation of the STING-TBK1-IRF3 pathway in DCs. Taken together, our findings suggest that Ad-IL15 is a candidate for cancer immunotherapy that promotes immune cell activation and infiltration, tumor vascular normalization and tertiary lymphoid structure formation in the TME., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793., (© 2022 by The American Society of Hematology.)

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that link plasma membrane proteins with actin filaments in the cell cortex. Hemizygous mutations in the X-linked moesin gene are associated with primary immunodeficiency with T and B cell lymphopenia, which also affects natural killer (NK) cells in most cases. We previously showed that moesin deficiency in mice substantially affects lymphocyte homeostasis, but its impact on NK cells remains unexplored. Here, we found that in moesin-deficient mice, NK cells were decreased in the peripheral blood and bone marrow but increased in the spleen. Analysis of female heterozygous mice showed a selective advantage for moesin-expressing NK cells in the blood. Moesin-deficient NK cells exhibited increased cell death and impaired signaling in response to IL-15, suggesting that moesin regulates NK cell survival through IL-15-mediated signaling. Our findings thus identify moesin as an NK cell homeostasis regulator in vivo., (Copyright © 2021 Elsevier Inc. All rights reserved.)

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days' culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vβ11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vβ11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vβ11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Interleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15., Results: IL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys., Conclusion: Immunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15., (© 2021. The Author(s).)

Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.

Zika virus (ZIKV) is an emerging arbovirus with recent global expansion. Historically, ZIKV infections with Asian lineages have been associated with mild disease such as rash and fever. However, recent Asian sub-lineages have caused outbreaks in the South Pacific and Latin America with increased prevalence of neurological disorders in infants and adults. Asian sub-lineage differences may partially explain the range of disease severity observed. However, the effect of Asian sub-lineage differences on pathogenesis remains poorly characterized. Current study conducts a head-to-head comparison of three Asian sub-lineages that are representative of the circulating ancestral mild Asian strain (ZIKV-SG), the 2007 epidemic French Polynesian strain (ZIKV-FP), and the 2013 epidemic Brazil strain (ZIKV-Brazil) in adult Cynomolgus macaques. Animals infected intervenously or subcutaneously with either of the three clinical isolates showed sub-lineage-specific differences in viral pathogenesis, early innate immune responses and systemic inflammation. Despite the lack of neurological symptoms in infected animals, the epidemiologically neurotropic ZIKV sub-lineages (ZIKV-Brazil and/or ZIKV-FP) were associated with more sustained viral replication, higher systemic inflammation (i.e. higher levels of TNFα, MCP-1, IL15 and G-CSF) and greater percentage of CD14+ monocytes and dendritic cells in blood. Multidimensional analysis showed clustering of ZIKV-SG away from ZIKV-Brazil and ZIKV-FP, further confirming sub-lineage differences in the measured parameters. These findings highlight greater systemic inflammation and monocyte recruitment as possible risk factors of adult ZIKV disease observed during the 2007 FP and 2013 Brazil epidemics. Future studies should explore the use of anti-inflammatory therapeutics as early treatment to prevent ZIKV-associated disease in adults.

Background: Glypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αβ T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αβ T cell therapy, including graft-versus-host disease (GvHD)., Methods: We developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR., Results: GPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity., Conclusions: Expanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors., Competing Interests: Competing interests: AM, XY, TB, AL, KPN, MMB, JTSW, SRYG, MTa, LB, AB, ZA, SA, BTA and MH are employees of Adicet Therapeutics. MTu is currently an employee of Allogene. DS is currently an employee of Astellas Pharma., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 μg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3 + CD8 + cytotoxic T lymphocytes, CD3 + CD5 dim CD21 - , and non-B/non-T NK cell populations, without stimulating T reg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rβ, and γ c ). IL-15 functions mainly via trans -presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the βγ c receptor-heterodimer on a neighboring T/NK cell. To date, no direct biophysical evidence for the intercellular assembly of the IL-15R heterotrimer exists. Ag presentation (AP), the initial step of T cell activation, is also based on APC-T cell interaction. We were compelled to ask whether AP has any effect on IL-15 TP or whether they are independent processes. In our human Raji B cell-Jurkat T cell model system, we monitored inter-/intracellular protein interactions upon formation of IL-15 TP and AP receptor complexes by Förster resonance energy transfer measurements. We detected enrichment of IL-15Rα and IL-2/15Rβ at the synapse and positive Förster resonance energy transfer efficiency if Raji cells were pretreated with IL-15, giving direct biophysical evidence for IL-15 TP. IL-15Rα and MHC class II interacted and translocated jointly to the immunological synapse when either ligand was present, whereas IL-2/15Rβ and CD3 moved independently of each other. IL-15 TP initiated STAT5 phosphorylation in Jurkat cells, which was not further enhanced by AP. Conversely, IL-15 treatment slightly attenuated Ag-induced phosphorylation of the CD3ζ chain. Our studies prove that in our model system, IL-15 TP and AP can occur independently, and although AP enhances IL-15R assembly, it has no significant effect on IL-15 signaling during TP. Thus, IL-15 TP can be considered an autonomous, Ag-independent process., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1 + tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8 + TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8 + TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8 + T cells, as depletion of CD8 + cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8 + and CD4 + TILs from human primary cancers in vitro , whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1 + TILs. See related Spotlight by Felices and Miller, p. 1110 ., (©2021 American Association for Cancer Research.)

IL-15, a proinflammatory cytokine critical for the generation, maintenance, and homeostasis of T cell responses, is produced naturally in response to HIV/SIV infection, but has also demonstrated therapeutic potential. IL-15 can boost CD4 + and CD8 + T cell and NK cell proliferation, activation, and function. However, IL-15 treatment may cause aberrant immune activation and accelerated disease progression in certain circumstances. Moreover, the relationship between the timing of IL-15 administration and disease progression remains unclear. The IL-15 superagonist N-803 was developed to expand the therapeutic potential of IL-15 by maximizing its tissue distribution and half-life. N-803 has garnered enthusiasm recently as a way to enhance the innate and cellular immune responses to HIV/SIV by improving CD8 + T cell recognition and killing of virus-infected cells and directing immune cells to mucosal sites and lymph nodes, the primary sites of virus replication. N-803 has also been evaluated in "shock and kill" strategies due to its potential to reverse latency (shock) and enhance antiviral immunity (kill). This review examines the current literature about the effects of IL-15 and N-803 on innate and cellular immunity, viral burden, and latency reversal in the context of HIV/SIV, and their therapeutic potential both alone and combined with additional interventions such as antiretroviral therapy (ART) and vaccination.

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Early responses to vaccination are important for shaping both humoral and cellular protective immunity. Dissecting innate vaccine signatures may predict immunogenicity to help optimize the efficacy of mRNA and other vaccine strategies. Here, we characterize the cytokine and chemokine responses to the 1 st and 2 nd dose of the BNT162b2 mRNA (Pfizer/BioNtech) vaccine in antigen-naive and in previously coronavirus disease 2019 (COVID-19)-infected individuals (NCT04743388). Transient increases in interleukin-15 (IL-15) and interferon gamma (IFN-γ) levels early after boost correlate with Spike antibody levels, supporting their use as biomarkers of effective humoral immunity development in response to vaccination. We identify a systemic signature including increases in IL-15, IFN-γ, and IP-10/CXCL10 after the 1 st vaccination, which were enriched by tumor necrosis factor alpha (TNF-α) and IL-6 after the 2 nd vaccination. In previously COVID-19-infected individuals, a single vaccination results in both strong cytokine induction and antibody titers similar to the ones observed upon booster vaccination in antigen-naive individuals, a result with potential implication for future public health recommendations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interindividual immune variability is driven predominantly by environmental factors, including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells-and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2 and IL-15 receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-γ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.

Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease that can progress to liver fibrosis. Recent clinical advance suggests a reversibility of liver fibrosis, but the cellular and molecular mechanisms underlying NASH resolution remain unclarified. Here, using a murine diet-induced NASH and the subsequent resolution model, we demonstrate direct roles of CD8 + tissue-resident memory CD8 + T (CD8 + Trm) cells in resolving liver fibrosis. Single-cell transcriptome analysis and FACS analysis revealed CD69 + CD103 - CD8 + Trm cell enrichment in NASH resolution livers. The reduction of liver CD8 + Trm cells, maintained by tissue IL-15, significantly delayed fibrosis resolution, while adoptive transfer of these cells protected mice from fibrosis progression. During resolution, CD8 + Trm cells attracted hepatic stellate cells (HSCs) in a CCR5-dependent manner, and predisposed activated HSCs to FasL-Fas-mediated apoptosis. Histological assessment of patients with NASH revealed CD69 + CD8 + Trm abundance in fibrotic areas, further supporting their roles in humans. These results highlight the undefined role of liver CD8 + Trm in fibrosis resolution., (© 2021. The Author(s).)

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly ( p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly ( p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher ( p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly ( p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly ( p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive ( p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Louis J. Picker and Scott G. Hansen declare their role as consultants for, and their substantial financial interest in Vir Biotechnology, Inc. as a Conflict of Interest that is managed by OHSU. No other authors have any conflicts to disclose.

HIV-specific CD8+ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4+ T cells from HIV+ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8+ T cell response have a profound impact on the emergence and persistence of escape mutations. This "participant-derived xenograft" model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies., Competing Interests: Disclosures: D.S. Jones reported "other" from Repertoire Immune Medicines and "other" from Bristol Myers Squibb outside the submitted work; in addition, D.S. Jones had a patent to PCT/US2019/061837 pending and a patent to PCT/US2017/037249 pending. T.L. Andresen reported being a co-founder of Repertoire Immune Medicine. C.M. Bollard reported "other" from Mana Therapeutics outside the submitted work; in addition, C.M. Bollard had a patent number 9,885,021 licensed to Mana Therapeutics. C.M. Bollard also reported being on the board of directors of Cabaletta Bio, being a co-founder of Mana Therapeutics and Catamaran Bio, and having stock ownership in Repertoire Immune Medicines and Neximmune. D.J. Irvine reported “other” from Repertoire Immune Medicines during the conduct of the study; in addition, D.J. Irvine had a patent to Cell Surface Coupling of Nanoparticles with royalties paid for Repertoire Immune Medicine. No other disclosures were reported., (© 2021 McCann et al.)

Background/aim: Recent studies have indicated that natural killer (NK) cells present in peripheral blood mononuclear cells (PBMCs) might be responsible for the somewhat poor outcome of clinical trials conducted with the NK cell line NK-92, as well as chimeric antigen receptor-modified NK-92 cells against leukemias and lymphomas. These NK cells and how their cytotoxic profiles can be altered by some common gamma chain receptor-dependent cytokines or by removal of CD4 + cells have been addressed herein., Materials and Methods: A time-resolved fluorometric assay using 2.2':6'.2"-terpyridine-6.6"-dicarboxylic acid-labeled NK-92 or K562 as target cells was used for measuring the cytotoxic activity of cytokine-treated PBMCs and purified NK cells., Results: Pre-incubation with 25 ng/ml interleukin 12 (IL-12), IL-15 or IL-21 for 72 h increased NK cell activity against K562 cells by more than 90% (1:25 target:effector ratio), whereas the corresponding NK cell activity against NK-92 cells was reduced by 15.9±0.1% by IL-12 and 50.6±2.9% by IL-15 compared to cells treated with medium alone. IL-7, on the other hand, increased NK activity against K562 to a much smaller extent (10.4±0.4%) and inhibited NK-92 cell lysis by 15.2±0.3%. Interestingly, similar amounts of IL-2 potentiated NK cell activity against both K562 and NK-92 cells by 50.9±0.5% and 14.3±0.9%, respectively. Purification of NK cells with magnetic beads demonstrated that NK cells indeed were responsible for the observed cytotoxic activity against both NK-92 cells (58.5±9.10%, 1:100 target:effector ratio) and K562 cells (81.6±9.57%, 1:100 target:effector ratio). Elimination of CD4+ cells from PBMCs did not alter the NK activity profile., Conclusion: This study highlights a problem that might arise with immune-based NK-92 and chimeric antigen receptor-transduced NK-92 cell therapies and pinpoints the need for evaluating new NK-like cell lines., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Group 3 innate lymphoid cells (ILC3s) in the gut mucosa have long been thought to be noncytotoxic lymphocytes that are critical for homeostasis of intestinal epithelial cells through secretion of IL-22. Recent work using human tonsillar cells demonstrated that ILC3s exposed to exogenous inflammatory cytokines for a long period of time acquired expression of granzyme B, suggesting that under pathological conditions ILC3s may become cytotoxic. We hypothesized that inflammation associated with bacterial exposure might trigger granzyme B expression in gut ILC3s. To test this, we exposed human colon lamina propria mononuclear cells to a panel of enteric bacteria. We found that the Gram-negative commensal and pathogenic bacteria induced granzyme B expression in a subset of ILC3s that were distinct from IL-22-producing ILC3s. A fraction of granzyme B + ILC3s coexpressed the cytolytic protein perforin. Granzyme B expression was mediated, in part, by IL-15 produced upon exposure to bacteria. ILC3s coexpressing all three IL-15R subunits (IL15Rα/β/γ) increased following bacterial stimulation, potentially allowing for cis presentation of IL-15 during bacterial exposure. Additionally, a large frequency of colonic myeloid dendritic cells expressed IL-15Rα, implicating myeloid dendritic cells in trans presentation of IL-15 to ILC3s. Tonsillar ILC3s minimally expressed granzyme B when exposed to the same bacteria or to rIL-15. Overall, these data establish the novel, to our knowledge, finding that human colonic ILC3s can express granzyme B in response to a subset of enteric bacteria through a process mediated by IL-15. These observations raise new questions about the multifunctional role of human gut ILC3s., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Differentially and functionally distinct T cell subsets are involved in the development of complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about factors regulating their recovery after HSCT. In this study, we investigated associations between immune-regulating cytokines, T cell differentiation, and clinical outcomes. We included 80 children undergoing allogeneic HSCT for acute leukemia using bone marrow or peripheral blood stem cells grafted from a matched sibling or unrelated donor. Cytokines (IL-7, IL-15, IL-18, SCF, IL-6, IL-2, and TNF-α) and active anti-thymocyte globulin (ATG) levels were longitudinally measured along with extended T cell phenotyping. The cytokine profiles showed a temporary rise in IL-7 and IL-15 during lymphopenia, which was strongly dependent on exposure to active ATG. High levels of IL-7 and IL-15 from graft infusion to day +30 were predictive of slower T cell recovery during the first 2 mo post-HSCT; however, because of a major expansion of memory T cell stages, only naive T cells remained decreased after 3 mo ( p < 0.05). No differential effect was seen on polarization of CD4 + T cells into Th1, Th2, or Th17 cells or regulatory T cells. Low levels of IL-7 and IL-15 at day +14 were associated with acute graft-versus-host disease grades II-IV in ATG-treated patients ( p = 0.0004 and p = 0.0002, respectively). Children with IL-7 levels comparable to healthy controls at day +14 post-HSCT were less likely to develop EBV reactivation posttransplant. These findings suggest that quantification of IL-7 and IL-15 may be useful as biomarkers in assessing the overall T cell depletion and suggest a potential for predicting complications after HSCT., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: Interleukin-15 (IL-15) is a critical cytokine for the development, proliferation, and function of natural killer (NK) cells, NKT cells, and CD8 + memory T cells and has become one of the most promising protein molecules for the treatment of cancer and viral diseases. However, there are several limitations in applying IL-15 in therapy, such as its low yield in vitro, limited potency, and short half-life in vivo. To date, there are several recombinant IL-15 agonists based on configurational modifications that are being pursued in the treatment of cancer, such as ALT-803, which are mainly produced from mammalian cells., Results: In this study, we designed two different forms of the IL-15 complex, which were formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor α (SuIL-15Rα)-IgG4 Fc fusion protein and designated IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc, respectively. The two IL-15 complexes were expressed in Pichia pastoris (P. pastoris), and their activities and half-lives were evaluated and compared. Pharmacokinetic analysis showed that IL-15/SuIL-15Rα-dFc had a half-life of 14.26 h while IL-15/SuIL-15Rα-mFc had a half-life of 9.16 h in mice, which were much longer than the 0.7-h half-life of commercial recombinant human IL-15 (rhIL-15). Treatment of mice with intravenous injection of the two IL-15 complexes resulted in significant increases in NK cells, NKT cells, and memory CD8 + T cells, which were not observed after rhIL-15 treatment. Treatment of human peripheral blood mononuclear cells (PBMCs) from healthy donors with the two IL-15 complexes yielded enhanced NK and CD8 + T cell activation and proliferation, which was comparable to the effect of rhIL-15., Conclusions: These findings indicate that the IL-15/SuIL-15Rα-dFc and IL-15/SuIL-15Rα-mFc produced in P. pastoris exhibit potent activities and prolonged half-lives and may serve as superagonists for immunotherapy in further research and applications.

The homeostasis of CD8 + CD122 + T cell requires IL-15 trans-presentation. We use Il15ra mutant mice and bone marrow chimeras to assess the role of IL-15 trans-presentation level in CD8 + CD122 + T cells homeostasis. We demonstrate that CD8 + CD122 + T cells require different levels of IL-15 trans-presentation to support their homeostasis., Competing Interests: Declaration of Competing Interest All contributing authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

The effects of local factors on activation of immune cells infiltrating the central nervous system (CNS) in a rat model of traumatic brain injury (TBI) remain elusive. The cytokine, interleukin (IL)-15, is crucial for development and activation of CD8 T lymphocytes, a prominent lymphocytic population present in TBI lesions. We investigated whether IL-15 originates from astrocytes and whether IL-15 can evoke the CD8 T-lymphocyte response in TBI. We observed that astrocytes were activated in a rat model of TBI and that IL-15 was overexpressed on the surface of astrocytes. Further, CD8 T lymphocytes infiltrating TBI lesions colocalized with IL-15-expressing astrocytes. Activated CD8 T lymphocytes released granzyme B (Gra-b), which, in turn, activated caspase-3-induced poly(ADP-ribose) polymerase cleavage and, ultimately, neuronal apoptosis. Conversely, inhibition of astrocyte activation by pre-treatment with the specific inhibitor, fluorocitrate (FC), that reduces carbon flux through the Krebs cycle in astrocytes resulted in improved neurological function and memory. FC pre-treatment was also associated with downregulated IL-15 expression and CD8 T-cell activation as well as decreased levels of neuronal apoptosis, suggesting that IL-15 initiated a domino effect toward apoptosis. In contrast, rats pre-treated with recombinant rat IL-15 showed upregulated CD8 T-cell numbers and Gra-b levels, in addition to induction of neuronal apoptosis. Together, our results indicated that IL-15 could induce neuronal apoptosis by enhancing CD8 T-cell function in a rat model of TBI.

T cell-based adoptive cell transfer therapy is now clinically used to fight cancer with CD19-targeting chimeric antigen receptor T cells. The use of other T cell-based immunotherapies relying on antigen-specific T cells, genetically modified or not, is expanding in various neoplastic diseases. T cell manufacturing has evolved through sophisticated processes to produce T cells with improved therapeutic potential. Clinical-grade manufacturing processes associated with these therapies must meet pharmaceutical requirements and therefore be standardized. Here, we focus on the use of cytokines to expand minimally differentiated T cells, as well as their standardization and harmonization in research and clinical settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8 + T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8 + T cell responses., Competing Interests: MP, NY and SD have ownership interest in patent filed for improved chimeric IL-15 [Indian patent number: E-2/2703/2017/MUM; App. Number: 201721010096 (published)]., (Copyright © 2021 Patidar, Yadav and Dalai.)

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer 1,2 . The accumulation of metabolites leads to cell stress and inflammation in the liver 3 , but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6 + CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6 + CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6 + CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor β chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1β induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1β to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1βR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.