Publications
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Ning, Jian-Fang; Davis, Zachary B; Zeb, Qudsia; Hinderlie, Peter; Tuininga, Katie & Wang, Liangjun
[Show all 20 contributors for this article]
(2026).
Induced pluripotent stem cell-derived natural killer cell therapy induces durable responses in glioblastoma and overcomes resistance via a B7-H3–targeted tri-specific killer engager.
Neuro-Oncology.
ISSN 1522-8517.
doi:
10.1093/neuonc/noag154.
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Abstract Background Glioblastoma is the most aggressive primary brain tumor, with a poor prognosis and limited treatment options. Natural killer (NK) cell therapy is a promising immunotherapeutic strategy; yet, its efficacy remains limited. We evaluated FT538, a clinical-grade NK product derived from induced pluripotent stem cells (iPSCs), in glioblastoma models. Methods FT538, engineered with a high-affinity noncleavable CD16 Fc receptor, a membrane-bound IL-15/IL-15Rα fusion protein, and CD38 knockout, was tested against 13 patient-derived glioblastoma stem-like cells (GSCs) in vitro and in orthotopic xenograft models. Intracranial persistence and neurotoxicity were assessed in mice. Surface proteomics identified therapeutic targets, and a B7-H3–targeted tri-specific killer engager (TriKE) was evaluated with FT538 and NKG2C+ adaptive NK cells. Results Glioblastoma stem-like cells were classified as sensitive (38%), moderately sensitive (38%), or resistant (23%) to FT538. Intracranial administration in mice was well tolerated, persisted for at least 35 days, and caused no neurotoxicity. A single intratumoral dose induced complete regression in sensitive xenografts. Surface profiling identified B7-H3 as a target to overcome resistance. Combination therapy with FT538 and a B7-H3 TriKE enhanced antitumor efficacy in resistant models, an effect also observed with adaptive NK cells. Conclusions FT538 exhibits potent tumoricidal activity in 77% of GSC lines (NK-sensitive and moderately sensitive), with curative potential in sensitive models, and demonstrates favorable persistence and tolerability in vivo. B7-H3–targeted TriKE restores NK sensitivity in resistant tumors. These findings provide a strong preclinical rationale for further clinical evaluation of FT538, alone or combined with B7-H3–targeted TriKE, for glioblastoma and other solid tumors.
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Rea, Anna; Santana-Hernández, Sara; Villanueva, Javier; Sanvicente-García, Marta; Cabo, Mariona & Suarez-Olmos, Jesús
[Show all 30 contributors for this article]
(2025).
Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression.
Nature Immunology.
ISSN 1529-2908.
26(4),
p. 582–594.
doi:
10.1038/s41590-025-02103-z.
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Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR–Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4KO NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4KO NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4KO conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.
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Vo, Dang Nghiem; Yuan, Ouyang; Kanaya, Minoru; Telliam-Dushime, Gladys; Li, Hongzhe & Kotova, Olga
[Show all 14 contributors for this article]
(2024).
A temporal developmental map separates human NK cells from noncytotoxic ILCs through clonal and single-cell analysis.
Blood Advances.
ISSN 2473-9529.
8(11),
p. 2933–2951.
doi:
10.1182/bloodadvances.2023011909.
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Natural killer (NK) cells represent the cytotoxic member within the innate lymphoid cell (ILC) family that are important against viral infections and cancer. Although the NK cell emergence from hematopoietic stem and progenitor cells through multiple intermediate stages and the underlying regulatory gene network has been extensively studied in mice, this process is not well characterized in humans. Here, using a temporal in vitro model to reconstruct the developmental trajectory of NK lineage, we identified an ILC-restricted oligopotent stage 3a CD34−CD117+CD161+CD45RA+CD56− progenitor population, that exclusively gave rise to CD56-expressing ILCs in vitro. We also further investigated a previously nonappreciated heterogeneity within the CD56+CD94−NKp44+ subset, phenotypically equivalent to stage 3b population containing both group-1 ILC and RORγt+ ILC3 cells, that could be further separated based on their differential expression of DNAM-1 and CD161 receptors. We confirmed that DNAM-1hi S3b and CD161hiCD117hi ILC3 populations distinctively differed in their expression of effector molecules, cytokine secretion, and cytotoxic activity. Furthermore, analysis of lineage output using DNA-barcode tracing across these stages supported a close developmental relationship between S3b-NK and S4-NK (CD56+CD94+) cells, whereas distant to the ILC3 subset. Cross-referencing gene signatures of culture-derived NK cells and other noncytotoxic ILCs with publicly available data sets validated that these in vitro stages highly resemble transcriptional profiles of respective in vivo ILC counterparts. Finally, by integrating RNA velocity and gene network analysis through single-cell regulatory network inference and clustering we unravel a network of coordinated and highly dynamic regulons driving the cytotoxic NK cell program, as a guide map for future studies on NK cell regulation.
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Fein, Joshua A.; Shouval, Roni; Krieger, Elizabeth; Spellman, Stephen R.; Wang, Tao & Baldauf, Henning
[Show all 19 contributors for this article]
(2024).
Systematic evaluation of donor-KIR/recipient-HLA interactions in HLA-matched hematopoietic cell transplantation for AML.
Blood Advances.
ISSN 2473-9529.
8(3),
p. 581–590.
doi:
10.1182/bloodadvances.2023011622.
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In acute myeloid leukemia (AML), donor natural killer cell killer immunoglobulin–like receptors (KIR) and recipient HLA interactions may contribute to the graft-versus-leukemia effect of allogeneic hematopoietic cell transplantation (HCT). Analyses of individual KIR/HLA interactions, however, have yielded conflicting findings, and their importance in the HLA-matched unrelated donor (MUD) setting remains controversial. We systematically studied outcomes of individual donor-KIR/recipient-HLA interactions for HCT outcomes and empirically evaluated prevalent KIR genotypes for clinical benefit. Adult patients with AML (n = 2025) who received HCT with MUD grafts in complete remission reported to the Center for International Blood and Marrow Transplantation were evaluated. Only the donor-2DL2+/recipient-HLA-C1+ pair was associated with reduced relapse (hazard ratio [HR], 0.79; 95% confidence interval [CI], 0.67-0.93; P = .006) compared with donor-2DL2–/recipient-HLA-C1+ pair. However, no association was found when comparing HLA-C groups among KIR-2DL2+–graft recipients. We identified 9 prevalent donor KIR genotypes in our cohort and screened them for association with relapse risk. Genotype 5 (G5) in all recipients and G3 in Bw4+ recipients were associated with decreased relapse risk (HR, 0.52; 95% CI, 0.35-0.78; P = .002; and HR, 0.32; 95% CI, 0.14-0.72; P = .006; respectively) and G2 (HR 1.63, 95% CI, 1.15-2.29; P = .005) with increased relapse risk in C1-homozygous recipients, compared with other patients with the same ligand. However, we could not validate these findings in an external data set of 796 AML transplants from the German transplantation registry. Neither a systematic evaluation of known HLA-KIR interactions nor an empiric assessment of prevalent KIR genotypes demonstrated clinically actionable associations; therefore, these data do not support these KIR-driven strategies for MUD selection in AML.
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Strunz, Benedikt; Momayyezi, Pouria; Bilev, Eleni; Muvva, Jagadeeswara Rao; Chen, Puran & Bister, Jonna
[Show all 74 contributors for this article]
(2024).
The HLA-B –21 M/T dimorphism associates with disease severity in COVID-19.
Genes and Immunity.
ISSN 1466-4879.
p. 1–5.
doi:
10.1038/s41435-024-00302-6.
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Host genetics shape immune responses and influence severity of infectious diseases. The HLA-B –21 M/T dimorphism tunes the functionality of natural killer (NK) cells expressing the inhibitory receptor NKG2A. NKG2A+ NK cells have been reported to recognize SARS-CoV-2-infected cells, but it remains unclear whether the HLA-B –21 M/T dimorphism associates with COVID-19 severity. Here, we investigated the influence of the HLA-B –21 M/T dimorphism in a cohort of 230 unvaccinated patients hospitalized with COVID-19 and requiring respiratory support. We found that HLA-B –21 M/M genotypes were more prevalent in patients with moderate compared to severe COVID-19 (6.0% vs. 0.9%). Comparison of age- and sex-matched sub-groups revealed that patients with M/M genotypes required mechanical respiratory support less frequently (OR = 0.13, 95% CI = 0.01-0.76, P = 0.013). Furthermore, patients with M/M genotypes showed a coordinately shifted signature of clinical laboratory parameters, coinciding with elevated serum levels of the anti-viral cytokine IFN-γ. These findings demonstrate that HLA-B variants associate with COVID-19 severity and suggest that the robust functionality of NKG2A+ NK cells in patients carrying the M/M genotype may contribute to protection from severe disease.
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Sætersmoen, Michelle Lu; Kotchetkov, Ivan S.; Torralba-Raga, Lamberto; Mansilla-Soto, Jorge; Sohlberg, Ebba & Krokeide, Silje Zandstra
[Show all 9 contributors for this article]
(2024).
Targeting HLA-E-overexpressing cancers with a NKG2A/C switch receptor.
Med.
ISSN 2666-6359.
p. 1–17.
doi:
10.1016/j.medj.2024.09.010.
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Background Human leukocyte antigen (HLA)-E is overexpressed by a large proportion of solid tumors, including malignant glioblastoma, and acts as a major checkpoint for NKG2A+ CD8+ T cells and natural killer (NK) cells in the tumor microenvironment and circulation. This axis operates alongside PD-L1 to inhibit effector responses by T and NK cells. Methods We engineered a chimeric A/C switch receptor, combining the high HLA-E binding affinity of the NKG2A receptor ectodomain with the activating signaling of the NKG2C receptor endodomain. The cytotoxic function of A/C switch-transduced NK and T cells was evaluated against tumor cells with varying levels of HLA-E expression. In vivo efficacy was assessed using a xenograft model of glioblastoma. Findings A/C switch-transduced NK and T cells exhibited superior and specific cytotoxicity against tumor cells with medium to high HLA-E expression. A/C switch-expressing human T cells demonstrated enhanced anti-tumor function in a glioblastoma xenograft model. The activity of the modified T cells was governed by an equilibrium between A/C switch levels and HLA-E expression, creating a therapeutic window to minimize on-target, off-tumor toxicities. Normal cells remained insensitive to A/C switch T cells, even after interferon (IFN)-γ pretreatment to induce HLA-E expression. Conclusions The A/C switch receptor effectively targets tumor cells expressing high levels of HLA-E, either alone or in combination with other engineered specificities, to overcome the suppressive NKG2A/HLA-E checkpoint. This approach offers a promising therapeutic strategy with a favorable safety profile for targeting HLA-E-overexpressing tumors. Funding This work was funded by The Research Council of Norway, the Norwegian Cancer Society, and the National Cancer Institute.
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Ask, Eivind Heggernes; Tschan-Plessl, Astrid; Hoel, Hanna Julie; Kolstad, Arne; Holte, Harald & Malmberg, Karl-Johan
(2024).
MetaGate: Interactive analysis of high-dimensional cytometry data with metadata integration.
Patterns.
5(7),
p. 1–12.
doi:
10.1016/j.patter.2024.100989.
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Flow cytometry is a powerful technology for high-throughput protein quantification at the single-cell level. Technical advances have substantially increased data complexity, but novel bioinformatical tools often show limitations in statistical testing, data sharing, cross-experiment comparability, or clinical data integration. We developed MetaGate as a platform for interactive statistical analysis and visualization of manually gated high-dimensional cytometry data with integration of metadata. MetaGate provides a data reduction algorithm based on a combinatorial gating system that produces a small, portable, and standardized data file. This is subsequently used to produce figures and statistical analyses through a fast web-based user interface. We demonstrate the utility of MetaGate through a comprehensive mass cytometry analysis of peripheral blood immune cells from 28 patients with diffuse large B cell lymphoma along with 17 healthy controls. Through MetaGate analysis, our study identifies key immune cell population changes associated with disease progression.
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Weiss, Lea; Schluck, Marjolein; Classens, René; Jonge, Paul K.J.D. de; Waart, Anniek van der & Nguyen, Khue G.
[Show all 13 contributors for this article]
(2024).
Interleukin-12 decorated nanosized semiflexible Immunofilaments enable directed targeting and augmented IFNγ responses of natural killer cells.
Acta Biomaterialia.
ISSN 1742-7061.
p. 386–397.
doi:
10.1016/j.actbio.2024.11.012.
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Immunotherapies are a powerful strategy to treat cancer by modulating the immune system to raise an anti-tumor immune response. A prime example of immunotherapies are cytokines - small immunomodulatory molecules that are widely used to stimulate immune cells. Undirected administration of cytokines, however, can cause severe side effects, preventing the use of potent cytokines, such as Interleukin (IL)-12, which induces IFNγ responses by cytotoxic effector lymphocytes, including NK cells. Biomaterials, like nanoparticles, can encapsulate IL-12 and accumulate at the tumor site to alleviate side effects. Yet, the released IL-12 might not be directly targeted to extracellular IL-12 receptors on the specific effector cells, thereby potentially compromising the cytokine's therapeutic efficacy. Here, we develop a polymer-based platform to target NK cells, which we call immunofilaments. Immunofilaments are nanosized linear polymers that present an anti-CD16 antibody and IL-12 effectively to NK cells and lead to synergistic NK cell activation as highlighted by an increase in TNFα and IFNγ production and upregulation of multiple activation markers, including CD25, CD69, and degranulation marker CD107a. NK cell proliferation is enhanced in the presence of both anti-CD16 antibody and IL-12 compared to giving IL-12 separately. Finally, we demonstrate that the IF platform is suitable for in vivo applications, as immunofilaments readily activate human NK cells upon administration to mice.
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Netskar, Herman Krogstad; Pfefferle, Aline; Goodridge, Jodie; Sohlberg, Ebba; Dufva, Olli & Teichmann, Sarah A
[Show all 12 contributors for this article]
(2024).
Pan-cancer profiling of tumor-infiltrating natural killer cells through transcriptional reference mapping.
Nature Immunology.
ISSN 1529-2908.
25,
p. 1445–1459.
doi:
10.1038/s41590-024-01884-z.
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The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic, receptor–ligand interactions and adaptive-like responses to viral infections. In the present study, we generated a single-cell transcriptional reference map of healthy human blood- and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene-regulatory networks defining NK cell differentiation. Transfer learning facilitated incorporation of tumor-infiltrating NK cell transcriptomes (39 datasets, 7 solid tumors, 427 patients) into the reference map to analyze tumor microenvironment (TME)-induced perturbations. Of the six functionally distinct NK cell states identified, a dysfunctional stressed CD56bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant effector CD56dim state were commonly enriched across tumor types, the ratio of which was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of new NK cell therapies and can be extended through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.
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Martin, Karen Elizabeth; Hammer, Quirin; Perica, Karlo; Sadelain, Michel & Malmberg, Karl-Johan
(2024).
Engineering immune-evasive allogeneic cellular immunotherapies.
Nature reviews. Immunology.
ISSN 1474-1733.
24,
p. 680–693.
doi:
10.1038/s41577-024-01022-8.
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Rebuffet, Lucas; Melsen, Janine E; Escaliere, Bertrand; Basurto-Lozada, Daniela; Bhandoola, Avinash & Björkström, Niklas K
[Show all 27 contributors for this article]
(2024).
High-dimensional single-cell analysis of human natural killer cell heterogeneity.
Nature Immunology.
ISSN 1529-2908.
25(8),
p. 1474–1488.
doi:
10.1038/s41590-024-01883-0.
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Natural killer (NK) cells are innate lymphoid cells (ILCs) contributing to immune responses to microbes and tumors. Historically, their classification hinged on a limited array of surface protein markers. Here, we used single-cell RNA sequencing (scRNA-seq) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to dissect the heterogeneity of NK cells. We identified three prominent NK cell subsets in healthy human blood: NK1, NK2 and NK3, further differentiated into six distinct subgroups. Our findings delineate the molecular characteristics, key transcription factors, biological functions, metabolic traits and cytokine responses of each subgroup. These data also suggest two separate ontogenetic origins for NK cells, leading to divergent transcriptional trajectories. Furthermore, we analyzed the distribution of NK cell subsets in the lung, tonsils and intraepithelial lymphocytes isolated from healthy individuals and in 22 tumor types. This standardized terminology aims at fostering clarity and consistency in future research, thereby improving cross-study comparisons.
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Hammer, Quirin; Perica, Karlo; Mbofung, Rina M.; Ooijen, Hanna van; Martin, Karen Elizabeth & Momayyezi, Pouria
[Show all 19 contributors for this article]
(2024).
Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies.
Cell Stem Cell.
ISSN 1934-5909.
31,
p. 1376–1386.
doi:
10.1016/j.stem.2024.06.011.
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Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.
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Pfefferle, Aline; Phuyal, Santosh; Netskar, Herman Krogstad & Malmberg, Karl-Johan
(2024).
Egr2 to the rescue: nanoparticles revitalize natural killer cells in the fight against cancer.
EMBO Journal.
ISSN 0261-4189.
43,
p. 2527–2529.
doi:
10.1038/s44318-024-00144-y.
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Reversal of tumor-induced natural killer (NK) cell dysfunction remains a major hurdle to overcome for the clinical success of immunotherapies based on NK cells targeting solid tumors. New research in The EMBO Journal identifies the transcription factor early growth response gene 2 (Egr2) and diacylglycerol kinase DGKα as two negative regulators of NK cell dysfunction (Sabag et al, 2024).
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Haroun-Izquierdo, Alvaro; Lanuza, Pilar M.; Pfefferle, Aline; Netskar, Herman Krogstad; Ask, Eivind Heggernes & Törlén, Johan
[Show all 9 contributors for this article]
(2023).
Effect of mTOR Inhibition with Sirolimus on Natural Killer Cell Reconstitution in Allogeneic Stem Cell Transplantation.
Transplantation and Cellular Therapy (TCT).
ISSN 2666-6375.
29(6),
p. 376.e1–376.e11.
doi:
10.1016/j.jtct.2023.03.023.
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Sirolimus is an inhibitor of the mammalian target of rapamycin (mTOR) and is emerging as a promising component of graft-versus-host disease (GVHD) prophylaxis regimens in the context of allogeneic hematopoietic stem cell transplantation (HSCT). Multiple studies have explored the clinical benefits of adding sirolimus to GVHD prophylaxis; however, detailed immunologic studies have not yet been carried out in this context. Mechanistically, mTOR is at the center of metabolic regulation in T cells and natural killer (NK) cells and is critical for their differentiation to mature effector cells. Therefore, close evaluation of the inhibition of mTOR in the context of immune reconstitution post-HSCT is warranted. In this work, we studied the effect of sirolimus on immune reconstitution using a biobank of longitudinal samples from patients receiving either tacrolimus/sirolimus (TAC/SIR) or cyclosporin A/methotrexate (CSA/MTX) as conventional GVHD prophylaxis. Healthy donor controls, donor graft material, and samples from 28 patients (14 with TAC/SIR, 14 with CSA/MTX) at 3 to 4 weeks and 34 to 39 weeks post- HSCT were collected. Multicolor flow cytometry was used to perform broad immune cell mapping, with a focus on NK cells. NK cell proliferation was evaluated over a 6-day in vitro homeostatic proliferation protocol. Furthermore, in vitro NK cell responses to cytokine stimulation or tumor cells were evaluated. Systems-level assessment of the immune repertoire revealed a deep and prolonged suppression (weeks 34 to 39 post-HSCT) of the naïve CD4 T cell compartment with relative sparing of regulatory T cells and enrichment of CD69+Ki-67+HLA-DR+ CD8 T cells, independent of the type of GVHD prophylaxis. Early after transplantation (weeks 3 to 4), while patients were still on TAC/SIR or CSA/MTX, we found a relative increase in less-differentiated CD56bright NK cells and NKG2A+CD57−KIR− CD56dim NK cells and a distinct loss of CD16 and DNAM-1 expression. Both regimens led to suppressed proliferative responses ex vivo and functional impairment with preferential loss of cytokine responsiveness and IFN-γ production. Patients who received TAC/SIR as GVHD prophylaxis showed delayed NK cell reconstitution with lower overall NK cell counts and fewer CD56bright and NKG2A+ CD56dim NK cells. Treatment with sirolimus-containing regimens generated similar immune cell profiles as conventional prophylaxis; however, the NK cell compartment seemed to be composed of slightly more mature NK cells. These effects were also present after the completion of GVHD prophylaxis, suggesting that mTOR inhibition with sirolimus leaves a lasting imprint on homeostatic proliferation and NK cell reconstitution following HSCT.
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Clement, Dennis; Szabo, Edina Krisztina; Krokeide, Silje Zandstra; Wiiger, Merete Thune; Vincenti, Marianna & Orellana, Daniel Alfredo Palacios
[Show all 13 contributors for this article]
(2023).
The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.
Journal of Immunology.
ISSN 0022-1767.
211(9),
p. 1348–1358.
doi:
10.4049/jimmunol.2300406.
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Abstract Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1−/− NK92 (NK92ML1−/−) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1−/− cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.
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Humbert, Marion; Olofsson, Anna; Wullimann, David; Niessl, Julia; Hodcroft, Emma B. & Cai, Curtis
[Show all 20 contributors for this article]
(2023).
Functional SARS-CoV-2 cross-reactive CD4<sup>+</sup> T cells established in early childhood decline with age.
Proceedings of the National Academy of Sciences of the United States of America (PNAS).
ISSN 0027-8424.
120(12).
doi:
10.1073/pnas.2220320120.
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Significance Our results provide evidence that functional pre-existing SARS-CoV-2-reactive memory CD4+ T cells are elicited in early childhood and linked to seroconversion with the seasonal coronavirus OC43 but not many other viral infections. Compared to other viruses, the high OC43 seroprevalence at age two indicates that memory responses to coronaviruses develop at a young age. The distinct age-dependent profiles of the responding T cells suggest that cross-reactive T cells can contribute to the different clinical outcomes of COVID-19 in children and the elderly. The present results provide important advances regarding antigen-specific memory CD4+ T cell development and maturation, which can help guide future vaccine and therapeutic interventions relating to specificity, function, and phenotype of memory T cell responses throughout the human life span. Abstract Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (β-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein–Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.
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Giang, Kim Anh; Boxaspen, Thorstein; Diao, Yumei; Nilvebrant, Johan; Kosugi-Kanaya, Mizuha & Kanaya, Minoru
[Show all 11 contributors for this article]
(2023).
Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells.
New Biotechnology.
ISSN 1871-6784.
77,
p. 139–148.
doi:
10.1016/j.nbt.2023.09.002.
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We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
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Philippon, Camille; Tao, Sudan; Clement, Dennis; Haroun-Izquierdo, Alvaro; Kichula, Katherine M. & Netskar, Herman Krogstad
[Show all 21 contributors for this article]
(2023).
Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires.
Blood Advances.
ISSN 2473-9529.
7(16),
p. 4492–4504.
doi:
10.1182/bloodadvances.2023009827.
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Abstract The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex–deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.
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Sandoz, Patrick A.; Kuhnigk, Kyra; Szabo, Edina Krisztina; Thunberg, Sarah; Erikson, Elina & Sandström, Niklas
[Show all 14 contributors for this article]
(2023).
Modulation of lytic molecules restrain serial killing in γδ T lymphocytes.
Nature Communications.
14(1),
p. 1–16.
doi:
10.1038/s41467-023-41634-7.
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Abstract γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies.
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Lachota, Mieszko; Zielniok, Katarzyna; Orellana, Daniel Alfredo Palacios; Kanaya, Minoru; Peena, Leena & Hoel, Hanna Julie
[Show all 11 contributors for this article]
(2023).
Mapping the chemotactic landscape in NK cells reveals subset-specific synergistic migratory responses to dual chemokine receptor ligation.
EBioMedicine.
96.
doi:
10.1016/j.ebiom.2023.104811.
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Background Natural killer (NK) cells have a unique capability of spontaneous cytotoxicity against malignant cells and hold promise for off-the-shelf cell therapy against cancer. One of the key challenges in the field is to improve NK cell homing to solid tumors. Methods To gain a deeper understanding of the cellular mechanisms regulating trafficking of NK cells into the tumor, we used high-dimensional flow cytometry, mass cytometry, and single-cell RNA-sequencing combined with functional assays, creating a comprehensive map of human NK cell migration phenotypes. Findings We found that the chemokine receptor repertoire of peripheral blood NK cells changes in a coordinated manner becoming progressively more diversified during NK cell differentiation and correlating tightly with the migratory response of the distinct NK cell subsets. Simultaneous ligation of CXCR1/2 and CX3CR1, synergistically potentiated the migratory response of NK cells. Analysis of 9471 solid cancers from publicly available TCGA/TARGET repositories revealed dominant chemokine patterns that varied across tumor types but with no tumor group expressing ligands for more than one chemokine receptor present on mature NK cells. Interpretation The finding that chemokine stimulation can elicit a synergistic migratory response in NK cells combined with the identified lack of naturally occurring pairs of chemokines-chemokine receptors in human cancers may explain the systematic exclusion of NK cells from the tumor microenvironment and provides a basis for engineering next-generation NK cell therapies against malignancies. Funding The Polish Ministry of Science and Higher Education, the National Science Centre, Poland, The Norwegian Cancer Society, the Norwegian Research Council, the South-Eastern Norway Regional Health Authority, The Swedish Cancer Society, the Swedish Children's Cancer Foundation, The Swedish Research Council, The Center of Excellence: Precision Immunotherapy Alliance, Knut and Alice Wallenberg Foundation and National Cancer Institute.
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Bajor, Malgorzata; Graczyk-Jarzynka, Agnieszka; Marhelava, Katsiaryna; Burdzinska, Anna; Muchowicz, Angelika & Goral, Agnieszka
[Show all 17 contributors for this article]
(2022).
PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells.
Journal for ImmunoTherapy of Cancer (JITC).
10(1).
doi:
10.1136/jitc-2021-002500.
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Background Immune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets. Methods New atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1–CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2–CAR T cells were used for combination with PD-L1–CAR T cells against MCF-7 cells. Results PD-L1–CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1–CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1–CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1–CAR cells. Accordingly, PD-L1–CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1–CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2–CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1–CAR T cells when used in combination. Conclusions In summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1–CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1–CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1–CAR strategy into clinical studies.
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Shapiro, Roman M.; Birch, Grace C.; Hu, Guangan; Cadavid, Juliana Vergara; Nikiforow, Sarah & Baginska, Joanna
[Show all 32 contributors for this article]
(2022).
Expansion, persistence, and efficacy of donor memory-like NK cells infused for posttransplant relapse.
Journal of Clinical Investigation.
ISSN 0021-9738.
132(11).
doi:
10.1172/JCI154334.
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Background. Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse. Methods. We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion. Results. In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. Conclusion. Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.
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Ljunggren, Hans-Gustaf; Ask, Eivind Heggernes; Cornillet, Martin; Strunz, Benedikt; Chen, Puran & Muvva, Jagadeeswara Rao
[Show all 42 contributors for this article]
(2022).
The Karolinska KI/K COVID-19 immune atlas: An open resource for immunological research and educational purposes.
Scandinavian Journal of Immunology.
ISSN 0300-9475.
96(1).
doi:
10.1111/sji.13195.
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The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allows scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.
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Momayyezi, Pouria; Malmberg, Karl-Johan & Hammer, Quirin
(2022).
Small Interfering RNA Delivery Into Primary Human Natural Killer Cells for Functional Gene Analyses.
Current Protocols.
2(11).
doi:
10.1002/cpz1.613.
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Orellana, Daniel Alfredo Palacios; Momayyezi, Pouria; Huhn, Oisín; Ask, Eivind Heggernes; Dunst, Josefine & Malmberg, Karl-Johan
[Show all 7 contributors for this article]
(2022).
An optimized platform for efficient siRNA delivery into human NK cells.
European Journal of Immunology.
ISSN 0014-2980.
doi:
10.1002/eji.202149710.
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The molecular networks that regulate natural killer (NK) cell functions are not completely understood. Here, we present a workflow for efficient delivery of siRNA into human NK cells without compromising viability. This methodology represents a promising approach for rapidly interrogating gene functions in primary human NK cells.
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Haroun-Izquierdo, Alvaro; Vincenti, Marianna; Netskar, Herman Krogstad; Ooijen, Hanna van; Zhang, Bin & Bendzick, Laura
[Show all 30 contributors for this article]
(2022).
Adaptive single-KIR+NKG2C+ NK cells expanded from select superdonors show potent missing-self reactivity and efficiently control HLA-mismatched acute myeloid leukemia.
Journal for ImmunoTherapy of Cancer (JITC).
10:e005577(11),
p. 1–13.
doi:
10.1136/jitc-2022-005577.
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Background Natural killer (NK) cells hold great promise as a source for allogeneic cell therapy against hematological malignancies, including acute myeloid leukemia (AML). Current treatments are hampered by variability in NK cell subset responses, a limitation which could be circumvented by specific expansion of highly potent single killer immunoglobulin-like receptor (KIR)+NKG2C+ adaptive NK cells to maximize missing-self reactivity. Methods We developed a GMP-compliant protocol to expand adaptive NK cells from cryopreserved cells derived from select third-party superdonors, that is, donors harboring large adaptive NK cell subsets with desired KIR specificities at baseline. We studied the adaptive state of the cell product (ADAPT-NK) by flow cytometry and mass cytometry as well as cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). We investigated the functional responses of ADAPT-NK cells against a wide range of tumor target cell lines and primary AML samples using flow cytometry and IncuCyte as well as in a mouse model of AML. Results ADAPT-NK cells were >90% pure with a homogeneous expression of a single self-HLA specific KIR and expanded a median of 470-fold. The ADAPT-NK cells largely retained their adaptive transcriptional signature with activation of effector programs without signs of exhaustion. ADAPT-NK cells showed high degranulation capacity and efficient killing of HLA-C/KIR mismatched tumor cell lines as well as primary leukemic blasts from AML patients. Finally, the expanded adaptive NK cells had preserved robust antibody-dependent cellular cytotoxicity potential and combination of ADAPT-NK cells with an anti-CD16/IL-15/anti-CD33 tri-specific engager led to near-complete killing of resistant CD45dim blast subtypes. Conclusions These preclinical data demonstrate the feasibility of off-the-shelf therapy with a non-engineered, yet highly specific, NK cell population with full missing-self recognition capability.
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Hammer, Quirin; Dunst, Josefine; Christ, Wanda; Picarazzi, Francesca; Wendorff, Mareike & Momayyezi, Pouria
[Show all 51 contributors for this article]
(2022).
SARS-CoV-2 Nsp13 encodes for an HLA-E-stabilizing peptide that abrogates inhibition of NKG2A-expressing NK cells.
Cell reports.
38(10).
doi:
10.1016/j.celrep.2022.110503.
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Natural killer (NK) cells are innate immune cells that contribute to host defense against virus infections. NK cells respond to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and are activated in patients with acute coronavirus disease 2019 (COVID-19). However, by which mechanisms NK cells detect SARS-CoV-2-infected cells remains largely unknown. Here, we show that the Non-structural protein 13 of SARS-CoV-2 encodes for a peptide that is presented by human leukocyte antigen E (HLA-E). In contrast with self-peptides, the viral peptide prevents binding of HLA-E to the inhibitory receptor NKG2A, thereby rendering target cells susceptible to NK cell attack. In line with these observations, NKG2A-expressing NK cells are particularly activated in patients with COVID-19 and proficiently limit SARS-CoV-2 replication in infected lung epithelial cells in vitro. Thus, these data suggest that a viral peptide presented by HLA-E abrogates inhibition of NKG2A+ NK cells, resulting in missing self-recognition.
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Mazzarella, Luca; Enblad, Gunilla; Olweus, Johanna; Malmberg, Karl-Johan & Jerkeman, Mats
(2021).
Advances in immune therapies in hematological malignancies.
Journal of Internal Medicine.
ISSN 0954-6820.
p. 1–16.
doi:
10.1111/joim.13395.
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Immunotherapy in cancer takes advantage of the exquisite specificity, potency, and flexibility of the immune system to eliminate alien tumor cells. It involves strategies to activate the entire immune defense, by unlocking mechanisms developed by tumor cells to escape from surrounding immune cells, as well as engineered antibody and cellular therapies. What is important to note is that these are therapeutics with curative potential. The earliest example of immune therapy is allogeneic stem cell transplantation, introduced in 1957, which is still an important modality in hematology, most notably in myeloid malignancies. In this review, we discuss developmental trends of immunotherapy in hematological malignancies, focusing on some of the strategies that we believe will have the most impact on future clinical practice in this field. In particular, we delineate novel developments for therapies that have already been introduced into the clinic, such as immune checkpoint inhibition and chimeric antigen receptor T-cell therapies. Finally, we discuss the therapeutic potential of emerging strategies based on T-cell receptors and adoptive transfer of allogeneic natural killer cells.
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Sohlberg, Ebba; Pfefferle, Aline; Ask, Eivind Heggernes; Tschan-Plessl, Astrid; Jacobs, Benedikt Marcel & Netskar, Herman Krogstad
[Show all 16 contributors for this article]
(2021).
Perturbed NK Cell Homeostasis Associated with Disease Severity in Chronic Neutropenia.
Blood.
ISSN 0006-4971.
doi:
10.1182/blood.2021013233.
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Abstract Neutrophils have been thought to play a critical role in terminal differentiation of NK cells. Whether this effect is direct or a consequence of global immune changes with effects on NK-cell homeostasis remains unknown. In this study, we used high-resolution flow and mass cytometry to examine NK-cell repertoires in 64 patients with neutropenia and 27 healthy age- and sex-matched donors. A subgroup of patients with chronic neutropenia showed severely disrupted NK-cell homeostasis manifesting as increased frequencies of CD56bright NK cells and a lack of mature CD56dim NK cells. These immature NK-cell repertoires were characterized by expression of the proliferation/exhaustion markers Ki-67, Tim-3, and TIGIT and displayed blunted tumor target cell responses. Systems-level immune mapping revealed that the changes in immunophenotypes were confined to NK cells, leaving T-cell differentiation intact. RNA sequencing of NK cells from these patients showed upregulation of a network of genes, including TNFSF9, CENPF, MKI67, and TOP2A, associated with apoptosis and the cell cycle, but different from the conventional CD56bright signatures. Profiling of 249 plasma proteins showed a coordinated enrichment of pathways related to apoptosis and cell turnover, which correlated with immature NK-cell repertoires. Notably, most of these patients exhibited severe-grade neutropenia, suggesting that the profoundly altered NK-cell homeostasis was connected to the severity of their underlying etiology. Hence, although our data suggest that neutrophils are dispensable for NK-cell development and differentiation, some patients displayed a specific gap in the NK repertoire, associated with poor cytotoxic function and more severe disease manifestations.
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Woan, Karrune V.; Kim, Hansol; Bjordahl, Ryan; Davis, Zachary B.; Gaidarova, Svetlana & Goulding, John
[Show all 31 contributors for this article]
(2021).
Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy.
Cell Stem Cell.
ISSN 1934-5909.
28(12).
doi:
10.1016/j.stem.2021.08.013.
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Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.
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Lachota, Mieszko; Lennikov, Anton; Malmberg, Karl-Johan & Zagozdzon, Radoslaw
(2021).
Bioinformatic Analysis Reveals Central Role for Tumor-Infiltrating Immune Cells in Uveal Melanoma Progression.
Journal of Immunology Research.
ISSN 2314-8861.
2021.
doi:
10.1155/2021/9920234.
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Pfefferle, Aline; Jacobs, Benedikt Marcel; Haroun-Izquierdo, Alvaro; Kveberg, Lise; Sohlberg, Ebba & Malmberg, Karl-Johan
(2020).
Deciphering natural killer cell homeostasis.
Frontiers in Immunology.
11:812,
p. 1–11.
doi:
10.3389/fimmu.2020.00812.
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Natural killer (NK) cells have a central role within the innate immune system, eliminating virally infected, foreign and transformed cells through their natural cytotoxic capacity. Release of their cytotoxic granules is tightly controlled through the balance of a large repertoire of inhibitory and activating receptors, and it is the unique combination of these receptors expressed by individual cells that confers immense diversity both in phenotype and functionality. The diverse, yet unique, NK cell repertoire within an individual is surprisingly stable over time considering the constant renewal of these cells at steady state. Here we give an overview of NK cell differentiation and discuss metabolic requirements, intra-lineage plasticity and transcriptional reprogramming during IL-15-driven homeostatic proliferation. New insights into the regulation of NK cell differentiation and homeostasis could pave the way for the successful implementation of NK cell-based immunotherapy against cancer.
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Clement, Dennis; Goodridge, Jodie; Grimm, Christian; Patel, Sandip & Malmberg, Karl-Johan
(2020).
TRP channels as interior designers: Remodeling the endolysosomal compartment in natural killer cells.
Frontiers in Immunology.
11:753,
p. 1–15.
doi:
10.3389/fimmu.2020.00753.
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Cytotoxic lymphocytes, including natural killer (NK) cells and T cells are distinguished by their ability to eliminate target cells through release of secretory lysosomes. Conventional lysosomes and secretory lysosomes are part of the pleomorphic endolysosomal system and characterized by its highly dynamic nature. Several calcium-permeable TRP calcium channels play an essential role in endolysosomal calcium signaling to ensure proper function of these organelles. In NK cells, the expression of self MHC-specific inhibitory receptors dynamically tunes their secretory potential in a non-transcriptional, calcium-dependent manner. New insights suggest that TRPML1-mediated lysosomal calcium fluxes are tightly interconnected to NK cell functionality through modulation of granzyme B and perforin content of the secretory lysosome. Lysosomal TRP channels show a subset-specific expression pattern during NK differentiation, which is paralleled with gradually increased loading of effector molecules in secretory lysosomes. Methodological advances, including organellar patch-clamping, specific pharmacological modulators, and genetically-encoded calcium indicators open up new possibilities to investigate how TRP channels influence communication between intracellular organelles in immune cells. This review discusses our current understanding of lysosome biogenesis in NK cells with an emphasis on the TRP mucolipin family and the implications for NK cell functionality and cancer immunotherapy.
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Maucourant, Christopher; Filipovic, Iva; Ponzetta, Andrea; Aleman, Soo; Cornillet, Martin & Hertwig, Laura
[Show all 28 contributors for this article]
(2020).
Natural killer cell immunotypes related to COVID-19 disease severity.
Science immunology.
5:eabd6832(50),
p. 1–15.
doi:
10.1126/SCIIMMUNOL.ABD6832.
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Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56 bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.
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Goldenson, Benjamin H.; Zhu, Huang; Wang, YunZu Michele; Heragu, Naveen; Bernareggi, Davide & Ruiz-Cisneros, Alessa
[Show all 11 contributors for this article]
(2020).
Umbilical cord blood and iPSC-derived natural killer cells demonstrate key differences in cytotoxic activity and KIR profiles.
Frontiers in Immunology.
11,
p. 1–14.
doi:
10.3389/fimmu.2020.561553.
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Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56+ NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34+ hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.
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Jacobs, Benedikt Marcel; Schlögl, Sebastian; Strobl, Carolin Dorothea; Völkl, Simon; Stoll, Andrej & Mougiakakos, Dimitrios
[Show all 9 contributors for this article]
(2020).
The oncometabolite 5′-deoxy-5′-methylthioadenosine blocks multiple signaling pathways of NK cell activation.
Frontiers in Immunology.
11:2128,
p. 1–13.
doi:
10.3389/fimmu.2020.02128.
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Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5′-Deoxy-5′-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.
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Pfefferle, Aline; Jacobs, Benedikt Marcel; Netskar, Herman; Ask, Eivind Heggernes; Lorenz, Susanne & Clancy, Trevor
[Show all 9 contributors for this article]
(2019).
Intra-lineage plasticity and functional reprogramming maintain natural killer cell repertoire diversity.
Cell reports.
29(8),
p. 2284–2294.e4.
doi:
10.1016/j.celrep.2019.10.058.
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Natural killer (NK) cell repertoires are made up of phenotypically distinct subsets with different functional properties. The molecular programs involved in maintaining NK cell repertoire diversity under homeostatic conditions remain elusive. Here, we show that subset-specific NK cell proliferation kinetics correlate with mTOR activation, and global repertoire diversity is maintained through a high degree of intra-lineage subset plasticity during interleukin (IL)-15-driven homeostatic proliferation in vitro. Slowly cycling sorted KIR+CD56dim NK cells with an induced CD57 phenotype display increased functional potential associated with increased transcription of genes involved in adhesion and immune synapse formation. Rapidly cycling cells upregulate NKG2A, display a general loss of functionality, and a transcriptional signature associated with increased apoptosis/cellular stress, actin-remodeling, and nuclear factor κB (NF-κB) activation. These results shed light on the role of intra-lineage plasticity during NK cell homeostasis and suggest that the functional fate of the cell is tightly linked to the acquired phenotype and transcriptional reprogramming.
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Enqvist, Monika; Jacobs, Benedikt Marcel; Junlen, Henna R.; Schaffer, Marie; Melen, Christopher Michael & Friberg, Danielle
[Show all 8 contributors for this article]
(2019).
Systemic and Intra-Nodal Activation of NK Cells After Rituximab Monotherapy for Follicular Lymphoma.
Frontiers in Immunology.
10.
doi:
10.3389/fimmu.2019.02085.
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Monotherapy with the anti-CD20 monoclonal antibody rituximab can induce complete responses (CR) in patients with follicular lymphoma (FL). Resting FcRγIII+ (CD16+) natural killer (NK) cells respond strongly to rituximab-coated target cells in vitro. Yet, the contribution of NK cells in the therapeutic effect in vivo remains unknown. Here, we followed the NK cell repertoire dynamics in the lymph node and systemically during rituximab monotherapy in patients with FL. At baseline, NK cells in the tumor lymph node had a naïve phenotype albeit they were more differentiated than NK cells derived from control tonsils as determined by the frequency of CD56dim NK cells and the expression of killer cell immunoglobulin-like receptors (KIR), CD57 and CD16. Rituximab therapy induced a rapid drop in NK cell numbers coinciding with a relative increase in the frequency of Ki67+ NK cells both in the lymph node and peripheral blood. The Ki67+ NK cells had slightly increased expression of CD16, CD57 and higher levels of granzyme A and perforin. The in vivo activation of NK cells was paralleled by a temporary loss of in vitro functionality, primarily manifested as decreased IFNγ production in response to rituximab-coated targets. However, patients with pre-existing NKG2C+ adaptive NK cell subsets showed less Ki67 upregulation and were refractory to the loss of functionality. These data reveal variable imprints of rituximab monotherapy on the NK cell repertoire, which may depend on pre-existing repertoire diversity.
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Goodridge, Jodie; Jacobs, Benedikt Marcel; Sætersmoen, Michelle Lu; Clement, Dennis; Hammer, Quirin & Clancy, Trevor
[Show all 24 contributors for this article]
(2019).
Remodeling of secretory lysosomes during education tunes functional potential in NK cells.
Nature Communications.
10:14,
p. 1–15.
doi:
10.1038/s41467-019-08384-x.
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Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.
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Sætersmoen, Michelle Lu; Hammer, Quirin; Valamehr, Bahram; Kaufman, Dan S. & Malmberg, Karl-Johan
(2018).
Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells.
Seminars in Immunopathology.
ISSN 1863-2297.
41,
p. 59–68.
doi:
10.1007/s00281-018-0721-x.
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Cell therapy is emerging as a very promising therapeutic modality against cancer, spearheaded by the clinical success of chimeric antigen receptor (CAR) modified T cells for B cell malignancies. Currently, FDA-approved CAR-T cell products are based on engineering of autologous T cells harvested from the patient, typically using a central manufacturing facility for gene editing before the product can be delivered to the clinic and infused to the patients. For a broader implementation of advanced cell therapy and to reduce costs, it would be advantageous to use allogeneic “universal” cell therapy products that can be stored in cell banks and provided upon request, in a manner analogous to biopharmaceutical drug products. In this review, we outline a roadmap for development of off-the-shelf cell therapy based on natural killer (NK) cells derived from induced pluripotent stem cells (iPSCs). We discuss strategies to engineer iPSC-derived NK (iPSC-NK) cells for enhanced functional potential, persistence, and homing.
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Oei, Vincent Yi Sheng; Siernicka, Marta; Graczyk-Jarzynka, Agnieszka; Hoel, Hanna Julie; Yang, Weiwen & Orellana, Daniel Alfredo Palacios
[Show all 17 contributors for this article]
(2018).
Intrinsic functional potential of NK-Cell subsets constrains retargeting driven by chimeric antigen receptors.
Cancer immunology research.
ISSN 2326-6066.
6(4),
p. 467–480.
doi:
10.1158/2326-6066.CIR-17-0207.
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Natural killer (NK) cells hold potential as a source of allogeneic cytotoxic effector cells for chimeric antigen receptor (CAR)-mediated therapies. Here, we explored the feasibility of transfecting CAR-encoding mRNA into primary NK cells and investigated how the intrinsic potential of discrete NK-cell subsets affects retargeting efficiency. After screening five second- and third-generation anti-CD19 CAR constructs with different signaling domains and spacer regions, a third-generation CAR with the CH2-domain removed was selected based on its expression and functional profiles. Kinetics experiments revealed that CAR expression was optimal after 3 days of IL15 stimulation prior to transfection, consistently achieving over 80% expression. CAR-engineered NK cells acquired increased degranulation toward CD19+ targets, and maintained their intrinsic degranulation response toward CD19− K562 cells. The response of redirected NK-cell subsets against CD19+ targets was dependent on their intrinsic thresholds for activation determined through both differentiation and education by killer cell immunoglobulin-like receptors (KIR) and/or CD94/NKG2A binding to self HLA class I and HLA-E, respectively. Redirected primary NK cells were insensitive to inhibition through NKG2A/HLA-E interactions but remained sensitive to inhibition through KIR depending on the amount of HLA class I expressed on target cells. Adaptive NK cells, expressing NKG2C, CD57, and self-HLA–specific KIR(s), displayed superior ability to kill CD19+, HLA low, or mismatched tumor cells. These findings support the feasibility of primary allogeneic NK cells for CAR engineering and highlight a need to consider NK-cell diversity when optimizing efficacy of cancer immunotherapies based on CAR-expressing NK cells.
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Santis, Francesca De; Vecchio, Michele Del; Castagnoli, Lorenzo; Braud, Filippo De; Franceschini, Davide & Fucà, Giovanni
[Show all 18 contributors for this article]
(2018).
Innovative therapy, monoclonal antibodies, and beyond: Highlights from the eighth annual meeting.
Cytokine & growth factor reviews.
ISSN 1359-6101.
44,
p. 1–10.
doi:
10.1016/j.cytogfr.2018.10.005.
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The eighth annual conference of “Innovative therapy, monoclonal antibodies, and beyond” was held in Milan on Jan. 26, 2018, and hosted by Fondazione IRCCS–Istituto Nazionale dei Tumori (Fondazione IRCCS INT). The conference was divided into two main scientific sessions, of i) pre-clinical assays and novel biotargets, and ii) clinical translation, as well as a third session of presentations from young investigators, which focused on recent achievements within Fondazione IRCCS INT on immunotherapy and targeted therapies. Presentations in the first session addressed the issue of cancer immunotherapy activity with respect to tumor heterogeneity, with key topics addressing: 1) tumor heterogeneity and targeted therapy, with the definition of the evolutionary Index as an indicator of tumor heterogeneity in both space and time; 2) the analysis of cancer evolution, with the introduction of the TRACERx Consortium—a multi-million pound UK research project focused on non-small cell lung cancer (NSCLC); 3) the use of anti-estrogen agents to boost immune recognition of breast cancer cells; and 4) the high degree of functional plasticity within the NK cell repertoire, including the expansion of adaptive NK cells following viral challenges. The second session addressed: 1) the effectiveness of radiotherapy to enhance the proportion of patients responsive to immune-checkpoint blockers (ICBs); 2) the use of MDSC scores in selecting melanoma patients with high probability to be responsive to ICBs; and 3) the relevance of the gut microbiome as a predictive factor, and the potential of its perturbation in increasing the immune response rate to ICBs. Overall, a picture emerged of tumor heterogeneity as the main limitation that impairs the effectiveness of anti-cancer therapies. Thus, the choice of a specific therapy based on reproducible and selective predictive biomarkers is an urgent unmet clinical need that should be addressed in order to increase the proportion of long-term responding patients and to improve the sustainability of novel drugs.
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Malmberg, Karl-Johan; Sohlberg, Ebba; Goodridge, Jodie & Ljunggren, Hans-Gustaf
(2017).
Immune selection during tumor checkpoint inhibition therapy paves way for NK-cell "missing self" recognition.
Immunogenetics.
ISSN 0093-7711.
69(8-9),
p. 547–556.
doi:
10.1007/s00251-017-1011-9.
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Malmberg, Karl-Johan; Carlsten, Mattias; Björklund, Andreas; Sohlberg, Ebba; Bryceson, Yenan & Ljunggren, Hans-Gustaf
(2017).
Natural killer cell-mediated immunosurveillance of human cancer.
Seminars in Immunology.
ISSN 1044-5323.
31,
p. 20–29.
doi:
10.1016/j.smim.2017.08.002.
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Netskar, Herman Krogstad; Malmberg, Karl-Johan & Clancy, Trevor
(2025).
Single-Cell Mapping of Natural Killer Cell Diversity. Informing Strategies for Cancer Immunotherapy.
Universitetet i Oslo.
ISSN 9788234806414.
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Natural Killer (NK) cells play an important role in eliminating infected and transformed cells, including tumor cells. In recent years there has been an increased interest in using NK cells for cellular therapies against cancer. An increased understanding of the biology of NK cells and how different subsets and states of NK cells operate and respond to their environment can aid in efforts to improve NK cell-based therapies. The thesis provides a single-cell characterization of NK cells and established a transcriptional reference map of human NK cells. The reference map allowed for characterization of the perturbed states within tumor-infiltrating NK cells as these could be compared to the reference data. We identified six new states of NK cells, found across blood, tissues and tumors. Of particular interest, we identified a stressed state and a cytotoxic state, both of which were enriched across tumors. Analysis of overall survival of cancer patients showed these to be clinically relevant across different cancer types. The ratio of cytotoxic to stressed NK cells was positively associated with survival, highlighting the role NK cells can play as effectors against tumors. This might inform the design of NK cells for use in therapy. The thesis also provides an analysis of NK cells stimulated with IL-15 to capture the transcriptional profiles associated with NK subsets after stimulation. This data facilitated analysis of NK cells derived from induced pluripotent stem cells. These cells, known as iNK cells, is an important source of cells for the development of cellular therapies. The analysis provided in the thesis showed the iNK cells to exhibit an immature phenotype, but that they also display a cytotoxic profile. Genetically edited iNK cells were analyzed and compared to normal NK cells. This identified gaps in the transcriptional and regulatory repertoire of these cells which provides opportunities for further engineering to design better therapies in the future.
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Published
Feb. 14, 2012 1:41 PM
- Last modified
Jan. 24, 2025 2:09 PM
