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Article ; Online: Oyez, Oyez, Oyez!

Saunders, Philippa M / Brooks, Andrew G / Rossjohn, Jamie

2023 Volume 24, Issue 7, Page(s) 1052–1053

Language	English
Publishing date	2023-06-12
Publishing country	United States
Document type	Journal Article
ZDB-ID	2016987-5
ISSN	1529-2916 ; 1529-2908
ISSN (online)	1529-2916
ISSN	1529-2908
DOI	10.1038/s41590-023-01541-x
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Article ; Online: Structure of the murine CD94-NKG2A receptor in complex with Qa-1

MacLachlan, Bruce J / Sullivan, Lucy C / Brooks, Andrew G / Rossjohn, Jamie / Vivian, Julian P

The FEBS journal

2024 Volume 291, Issue 7, Page(s) 1530–1544

Abstract: The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell ... ...

Abstract	The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell surface expression levels through binding to MHC class Ia-derived leader sequence peptides presented by HLA class I histocompatibility antigen, alpha chain E (HLA-E; in humans) or H-2 class I histocompatibility antigen, D-37 (Qa-1
MeSH term(s)	Animals ; Humans ; Mice ; Histocompatibility Antigens Class I/genetics ; Histocompatibility Antigens Class I/metabolism ; HLA Antigens/genetics ; HLA Antigens/metabolism ; HLA-E Antigens ; Killer Cells, Natural ; NK Cell Lectin-Like Receptor Subfamily C/genetics ; NK Cell Lectin-Like Receptor Subfamily C/metabolism ; NK Cell Lectin-Like Receptor Subfamily D/genetics ; NK Cell Lectin-Like Receptor Subfamily D/chemistry ; Peptides/metabolism ; Protein Sorting Signals ; Receptors, Natural Killer Cell/metabolism
Chemical Substances	Histocompatibility Antigens Class I ; HLA Antigens ; HLA-E Antigens ; NK Cell Lectin-Like Receptor Subfamily C ; NK Cell Lectin-Like Receptor Subfamily D ; Peptides ; Protein Sorting Signals ; Receptors, Natural Killer Cell
Language	English
Publishing date	2024-01-10
Publishing country	England
Document type	Journal Article
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.17050
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Article ; Online: Examining the impact of immunosuppressive drugs on antibody-dependent cellular cytotoxicity (ADCC) of human peripheral blood natural killer (NK) cells and gamma delta (γδ) T cells.

Jalali, Sedigheh / Stankovic, Sanda / Westall, Glen P / Reading, Patrick C / Sullivan, Lucy C / Brooks, Andrew G

Transplant immunology

2023 Volume 82, Page(s) 101962

Abstract: Background: Human natural killer (NK) cells and gamma delta (γδ) T cells may impact outcomes of solid organ transplantation (SOT) such as lung transplantation (LTx) following the differential engagement of an array of activating and inhibitory receptors. ...

Abstract	Background: Human natural killer (NK) cells and gamma delta (γδ) T cells may impact outcomes of solid organ transplantation (SOT) such as lung transplantation (LTx) following the differential engagement of an array of activating and inhibitory receptors. Amongst these, CD16 may be particularly important due to its capacity to bind IgG to trigger antibody-dependent cellular cytotoxicity (ADCC) and the production of proinflammatory cytokines. While the use of immunosuppressive drugs (ISDs) is an integral component of SOT practice, their relative impact on various immune cells, especially γδT cells and CD16-induced functional responses, is still unclear. Methods: The ADCC responses of peripheral blood NK cells and γδT cells from both healthy blood donors and adult lung transplant recipients (LTRs) were assessed by flow cytometry. Specifically, the degranulation response, as reflected in the expression of CD107a, and the capacity of both NK cells and γδT cells to produce IFN-γ and TNF-α was assessed following rituximab (RTX)-induced activation. Additionally, the effect of cyclosporine A (CsA), tacrolimus (TAC), prednisolone (Prdl) and azathioprine (AZA) at the concentration of 1 ng/ml, 10 ng/ml, 100 ng/ml, and 1000 ng/ml on these responses was also compared in both cell types. Results: Flow cytometric analyses of CD16 expresion showed that its expression on γδT cells was both at lower levels and more variable than that on peripheral blood NK cells. Nevertheless functional analyses showed that despite these differences, γδT cells like NK cells can be readily activated by engagement with RTX to degranulate and produce cytokines such as IFNg and TNF-a. RTX-induced degranulation by either NK cells or γδT cells from healthy donors was not impacted by co-culture with individual ISDs. However, CsA and TAC but not Prdl and AZA did inhibit the production of IFN-γ and TNF-α by both cell types. Flow cytometric analyses of RTX-induced activation of NK cells and γδT cells from LTRs suggested their capacity to degranulate was not markedly impacted by transplantation with similar levels of cells expressing CD107 pre- and post-LTx. However an impairment in the ability of NK cells to produce cytokines was observed in samples obtained post LTx whereas γδT cell cytokine responses were not significantly impacted. Conclusions: In conclusion, the findings show that despite differences in the expression levels of CD16, γδT cells like NK cells can be readily activated by engagement with RTX and that in vitro exposure to CsA and TAC (calcineurin inhibitors) had a measurable effect on cytokine production but not degranulation by both NK cells and gdT cells from healthy donors. Finally the observation that in PBMC obtained from LTx recipients, NK cells but not γδT cells exhibited impaired cytokine reponses suggests that transplantation or chronic exposure to ISDs differentially impacts their potential to respond to the introduction of an allograft and/or transplant-associated infections.
MeSH term(s)	Adult ; Humans ; Tumor Necrosis Factor-alpha/metabolism ; Leukocytes, Mononuclear/metabolism ; Immunosuppressive Agents/therapeutic use ; Immunosuppressive Agents/pharmacology ; Killer Cells, Natural ; Antibody-Dependent Cell Cytotoxicity ; Cytokines/metabolism ; Cyclosporine/pharmacology ; Tacrolimus ; Prednisolone/pharmacology ; T-Lymphocytes/metabolism
Chemical Substances	Tumor Necrosis Factor-alpha ; Immunosuppressive Agents ; Cytokines ; Cyclosporine (83HN0GTJ6D) ; Tacrolimus (WM0HAQ4WNM) ; Prednisolone (9PHQ9Y1OLM)
Language	English
Publishing date	2023-11-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1160846-8
ISSN	1878-5492 ; 0966-3274
ISSN (online)	1878-5492
ISSN	0966-3274
DOI	10.1016/j.trim.2023.101962
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Article: Prediction of KIR3DL1/Human Leukocyte Antigen binding.

Maiers, Martin / Louzoun, Yoram / Pymm, Philip / Vivian, Julian P / Rossjohn, Jamie / Brooks, Andrew G / Saunders, Philippa M

bioRxiv : the preprint server for biology

2024

Abstract: KIR3DL1 is a polymorphic inhibitory Natural Killer (NK) cell receptor that recognizes Human Leukocyte Antigen (HLA) class I allotypes that contain the Bw4 motif. Structural analyses have shown that in addition to residues 77-83 that span the Bw4 motif, ... ...

Abstract	KIR3DL1 is a polymorphic inhibitory Natural Killer (NK) cell receptor that recognizes Human Leukocyte Antigen (HLA) class I allotypes that contain the Bw4 motif. Structural analyses have shown that in addition to residues 77-83 that span the Bw4 motif, polymorphism at other sites throughout the HLA molecule can influence the interaction with KIR3DL1. Given the extensive polymorphism of both KIR3DL1 and HLA class I, we built a machine learning prediction model to describe the influence of allotypic variation on the binding of KIR3DL1 to HLA class I. Nine KIR3DL1 tetramers were screened for reactivity against a panel of HLA class I molecules which revealed different patterns of specificity for each KIR3DL1 allotype. Separate models were trained for each of KIR3DL1 allotypes based on the full amino sequence of exons 2 and 3 encoding the
Language	English
Publishing date	2024-05-05
Publishing country	United States
Document type	Preprint
DOI	10.1101/2024.05.03.592082
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Article ; Online: Isoforms of Human MARCH1 Differ in Ability to Restrict Influenza A Viruses Due to Differences in Their N Terminal Cytoplasmic Domain.

Villalón-Letelier, Fernando / Farrukee, Rubaiyea / Londrigan, Sarah L / Brooks, Andrew G / Reading, Patrick C

Viruses

2022 Volume 14, Issue 11

Abstract: MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known ... ...

Abstract	MARCH1 and MARCH8 are closely related E3 ubiquitin ligases that ubiquitinate an overlapping spectrum of host proteins and restrict replication of certain viruses. While the antiviral activity of MARCH8 has been intensively studied, less is known regarding virus inhibition by MARCH1. Isoforms 1 and 2 of MARCH1 are very similar in overall structure but show major differences in their N-terminal cytoplasmic domain (N-CT). Herein, we used a doxycycline-inducible overexpression system to demonstrate that MARCH1.1 reduces titres of influenza A virus (IAV) released from infected cells whereas MARCH1.2 does not. The deletion of the entire N-CT of MARCH1.2 restored its ability to restrict IAV infectivity and sequential deletions mapped the restoration of IAV inhibition to delete the 16 N-terminal residues within the N-CT of MARCH1.2. While only MARCH1.1 mediated anti-IAV activity, qPCR demonstrated the preferential expression of MARCH1.2 over MARCH1.1 mRNA in unstimulated human peripheral blood mononuclear cells and also in monocyte-derived macrophages. Together, these studies describe the differential ability of MARCH1 isoforms to restrict IAV infectivity for the first time. Moreover, as published immunological, virological and biochemical studies examining the ability of MARCH1 to target particular ligands generally use only one of the two isoforms, these findings have broader implications for our understanding of how MARCH1 isoforms might differ in their ability to modulate particular host and/or viral proteins.
MeSH term(s)	Humans ; Influenza A virus/genetics ; Leukocytes, Mononuclear ; Protein Isoforms/genetics ; Influenza, Human ; Antiviral Agents
Chemical Substances	Protein Isoforms ; Antiviral Agents
Language	English
Publishing date	2022-11-18
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v14112549
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Article: Caveats of Using Overexpression Approaches to Screen Cellular Host IFITM Proteins for Antiviral Activity.

Meischel, Tina / Fritzlar, Svenja / Villalón-Letelier, Fernando / Smith, Jeffrey M / Brooks, Andrew G / Reading, Patrick C / Londrigan, Sarah L

Pathogens (Basel, Switzerland)

2023 Volume 12, Issue 4

Abstract: Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression ... ...

Abstract	Ectopic protein overexpression in immortalised cell lines is a commonly used method to screen host factors for their antiviral activity against different viruses. However, the question remains as to what extent such artificial protein overexpression recapitulates endogenous protein function. Previously, we used a doxycycline-inducible overexpression system, in conjunction with approaches to modulate the expression of endogenous protein, to demonstrate the antiviral activity of IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus-3 (PIV-3) in A549 cells. We now show that constitutive overexpression of the same IFITM constructs in A549 cells led to a significant restriction of PIV-3 infection by all three IFITM proteins. Variable IFITM mRNA and protein expression levels were detected in A549 cells with constitutive versus inducible overexpression of each IFITM. Our findings show that overexpression approaches can lead to levels of IFITM1, IFITM2, and IFITM3 that significantly exceed those achieved through interferon stimulation of endogenous protein. We propose that exceedingly high levels of overexpressed IFITMs may not accurately reflect the true function of endogenous protein, thus contributing to discrepancies when attributing the antiviral activity of individual IFITM proteins against different viruses. Our findings clearly highlight the caveats associated with overexpression approaches used to screen cellular host proteins for antiviral activity.
Language	English
Publishing date	2023-03-27
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens12040519
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Article ; Online: Influenza A Infection Stimulates RIG-I and Enhances Effector Function of Primary Human NK Cells.

Mohamed, Adham Abuelola / Soler, Sofía / Wegner, Julia / Bartok, Eva / Stankovic, Sanda / Brooks, Andrew G / Schlee, Martin

International journal of molecular sciences

2023 Volume 24, Issue 15

Abstract: Immune surveillance by natural killer (NK) cells and their recruitment to sites of inflammation renders them susceptible to viral infection, potentially modulating their effector function. Here, we analyzed innate RNA receptor signaling in NK cells ... ...

Abstract	Immune surveillance by natural killer (NK) cells and their recruitment to sites of inflammation renders them susceptible to viral infection, potentially modulating their effector function. Here, we analyzed innate RNA receptor signaling in NK cells downstream of direct Influenza A virus (IAV) infection and its impact on NK cell effector function. Infection of NK cells with IAV resulted in the activation of TBK1, NF-ϰB and subsequent type-I IFN secretion. CRISPR-generated knockouts in primary human NK cells revealed that this effect depended on the antiviral cytosolic RNA receptor RIG-I. Transfection of NK cells with synthetic 3p-dsRNA, a strong RIG-I agonist that mimics viral RNA, resulted in a similar phenotype and rendered NK cells resistant to subsequent IAV infection. Strikingly, both IAV infection and 3p-dsRNA transfection enhanced degranulation and cytokine production by NK cells when exposed to target cells. Thus, RIG-I activation in NK cells both supports their cell intrinsic viral defense and enhances their cytotoxic effector function against target cells.
MeSH term(s)	Humans ; Influenza A virus/physiology ; Influenza, Human ; Interferon Type I ; Killer Cells, Natural ; RNA
Chemical Substances	Interferon Type I ; RNA (63231-63-0) ; RIGI protein, human (EC 3.6.1.-)
Language	English
Publishing date	2023-07-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms241512220
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Article: TRIM16 Overexpression in HEK293T Cells Results in Cell Line-Specific Antiviral Activity.

Nigos, Lance R / Scott, Nichollas E / Brooks, Andrew G / Ait-Goughoulte, Malika / Londrigan, Sarah L / Reading, Patrick C / Farrukee, Rubaiyea

Pathogens (Basel, Switzerland)

2023 Volume 12, Issue 6

Abstract: Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of ... ...

Abstract	Host cell restriction factors are intracellular proteins that can inhibit virus replication. Characterisation of novel host cell restriction factors can provide potential targets for host-directed therapies. In this study, we aimed to assess a member of the Tripartite-motif family protein (TRIM) family, TRIM16, as a putative host cell restriction factor. To this end, we utilized constitutive or doxycycline-inducible systems to overexpress TRIM16 in HEK293T epithelial cells and then tested for its ability to inhibit growth by a range of RNA and DNA viruses. In HEK293T cells, overexpression of TRIM16 resulted in potent inhibition of multiple viruses, however, when TRIM16 was overexpressed in other epithelial cell lines (A549, Hela, or Hep2), virus inhibition was not observed. When investigating the antiviral activity of endogenous TRIM16, we report that siRNA-mediated knockdown of TRIM16 in A549 cells also modulated the mRNA expression of other TRIM proteins, complicating the interpretation of results using this method. Therefore, we used CRISPR/Cas9 editing to knockout TRIM16 in A549 cells and demonstrate that endogenous TRIM16 did not mediate antiviral activity against the viruses tested. Thus, while initial overexpression in HEK293T cells suggested that TRIM16 was a host cell restriction factor, alternative approaches did not validate these findings. These studies highlight the importance of multiple complementary experimental approaches, including overexpression analysis in multiple cell lines and investigation of the endogenous protein, when defining host cell restriction factors with novel antiviral activity.
Language	English
Publishing date	2023-06-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens12060852
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Article ; Online: Mouse guanylate-binding protein 1 does not mediate antiviral activity against influenza virus in vitro or in vivo.

Tessema, Melkamu B / Tuipulotu, Daniel Enosi / Oates, Clare V / Brooks, Andrew G / Man, Si Ming / Londrigan, Sarah L / Reading, Patrick C

Immunology and cell biology

2023 Volume 101, Issue 5, Page(s) 383–396

Abstract: Many interferon (IFN)-stimulated genes are upregulated within host cells following infection with influenza and other viruses. While the antiviral activity of some IFN-stimulated genes, such as the IFN-inducible GTPase myxoma resistance (Mx)1 protein 1, ... ...

Abstract	Many interferon (IFN)-stimulated genes are upregulated within host cells following infection with influenza and other viruses. While the antiviral activity of some IFN-stimulated genes, such as the IFN-inducible GTPase myxoma resistance (Mx)1 protein 1, has been well defined, less is known regarding the antiviral activities of related IFN-inducible GTPases of the guanylate-binding protein (GBP) family, particularly mouse GBPs, where mouse models can be used to assess their antiviral properties in vivo. Herein, we demonstrate that mouse GBP1 (mGBP1) was upregulated in a mouse airway epithelial cell line (LA-4 cells) following pretreatment with mouse IFNα or infection by influenza A virus (IAV). Whereas doxycycline-inducible expression of mouse Mx1 (mMx1) in LA-4 cells resulted in reduced susceptibility to IAV infection and reduced viral growth, inducible mGBP1 did not. Moreover, primary cells isolated from mGBP1-deficient mice (mGBP1
MeSH term(s)	Animals ; Humans ; Mice ; Antiviral Agents/metabolism ; Influenza A virus ; Influenza, Human/genetics ; Interferons/metabolism ; Macrophages/metabolism ; GTP-Binding Proteins/metabolism
Chemical Substances	Antiviral Agents ; Interferons (9008-11-1) ; GTP-Binding Proteins (EC 3.6.1.-)
Language	English
Publishing date	2023-02-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	284057-1
ISSN	1440-1711 ; 0818-9641
ISSN (online)	1440-1711
ISSN	0818-9641
DOI	10.1111/imcb.12627
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Article ; Online: Correction: DX5/CD49b-Positive T Cells Are Not Synonymous with CD1d-Dependent NKT Cells.

Pellicci, Daniel G / Hammond, Kirsten J L / Coquet, Jonathan / Kyparissoudis, Konstantinos / Brooks, Andrew G / Kedzierska, Katherine / Keating, Rachael / Turner, Stephen / Berzins, Stuart / Smyth, Mark J / Godfrey, Dale I

Journal of immunology (Baltimore, Md. : 1950)

2024 Volume 212, Issue 10, Page(s) 1603

Language	English
Publishing date	2024-03-27
Publishing country	United States
Document type	Published Erratum
ZDB-ID	3056-9
ISSN	1550-6606 ; 0022-1767 ; 1048-3233 ; 1047-7381
ISSN (online)	1550-6606
ISSN	0022-1767 ; 1048-3233 ; 1047-7381
DOI	10.4049/jimmunol.2400135
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