Phenotypic and functional characteristics of highly differentiated CD57 +NKG2C + NK cells in HIV-1- infected individuals

Natural killer (NK) cells are important anti-viral effector cells. The function and phenotype of the NK cells that constitute an individual’s NK cell repertoire can be influenced by ongoing and/or previous viral infections. Indeed, infection with human cytomegalovirus (HCMV) drives the expansion of a highly differentiated NK cell population characterized by expression of CD57 and the activating NKG2C receptor. This NK cell population has also been noted to occur in HIV-1-infected individuals. We evaluated the NK cells of HIV-1-infected and –uninfected individuals to determine the relative frequency of highly differentiated CD57 +NKG2C + NK cells and characterize these cells for their receptor expression and responsiveness to diverse stimuli. Highly differentiated CD57 +NKG2C + NK cells occurred at higher frequencies in HCMV-infected donors relative to HCMV-uninfected donors and were dramatically expanded in HIV-1/HCMV co-infected donors. The expanded CD57 +NKG2C + NK cell population in HIV-1-infected donors remained stable following antiretroviral therapy. CD57 +NKG2C + NK cells derived from HIV-1-infected individuals were robustly activated by antibody-dependent stimuli that contained anti-HIV-1 antibodies or therapeutic anti-CD20 antibody, and these NK cells mediated cytolysis through NKG2C. Lastly, CD57 +NKG2C + NK cells from HIV-1-infected donors were characterized by reduced expression of the inhibitory NKG2A receptor. The abundance of highly functional CD57 +NKG2C + NK cells in HIV-1-infected individuals raises the possibility that these NK cells could play a role in HIV-1 pathogenesis or serve as effector cells for therapeutic/cure strategies

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2−specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269–277 and A2/Orf1ab3183–3191. Using peptide−HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10−5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10−6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein–Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10−4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Influenza infection enhances antibody-mediated NK cell functions via Type I interferon dependent pathways

NK cells are an important component in the control of influenza infection, acting to both clear virus-infected cells and release antiviral cytokines. Engagement of CD16 on NK cells by antibody-coated influenza-infected cells results in antibody-dependent cellular cytotoxicity (ADCC). Increasing the potency of antibody-mediated NK cell activity could ultimately lead to improved control of influenza infection. To understand if NK cells can be functionally enhanced following exposure to influenza virus-infected cells, we co-cultured human PBMCs with influenza-infected human alveolar epithelial (A549) cells and evaluated the capacity of NK cells to mediate antibody-dependent functions. Pre-incubation of PBMCs with influenza-infected cells markedly enhanced the ability of NK cells to respond to immune complexes containing HA and anti-HA antibodies or transformed allogenic cells in the presence or absence of a therapeutic monoclonal antibody. Cytokine multiplex, RNA sequencing, supernatant transfer, trans-well and cytokine blocking/supplementation experiments showed that type I interferons released from PBMCs were primarily responsible for the influenza-induced enhancement of antibody-mediated NK cell functions. Importantly, the influenza-mediated increase in antibody-dependent NK cell functionality was mimicked by the type I interferon agonist poly(I:C). We conclude that type I interferon secretion induced by influenza virus infection enhances the capacity of NK cells to mediate ADCC, and this pathway could be manipulated to alter the potency of anti-influenza therapies and vaccines

Mucosal-associated invariant T cells are depleted and exhibit altered chemokine receptor expression and elevated granulocyte macrophage-colony stimulating factor production during end-stage renal disease

Background: End-stage renal disease (ESRD) is associated with an increased susceptibility to infectious diseases, including infection with Mycobacterium tuberculosis (Mtb). Mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites produced by many bacterial species, including Mtb, and may play an important role in providing protective immunity against tuberculosis infection in the lung. To date, little is known about MAIT cell frequency, phenotype, or function in ESRD patients. Methods: MAIT cells, identified by surface marker expression or MR1 tetramer binding, were characterized in 20 ESRD and 20 healthy control participants by multicolor flow cytometry. Ex vivo MAIT cell phenotype and cytokine production following PMA/ionomycin, IL-12/IL-18, or Escherichia coli stimulation were determined. Monocyte phenotype and plasma C-reactive protein/inflammatory cytokine levels were quantified by flow cytometry, ELISA, and multiplex bead array. Results: Peripheral blood MAIT cells were significantly depleted among ESRD patients compared to controls by both phenotypic and tetramer analysis and exhibited a loss of CXCR3 expression coupled to increased expression of CCR6 and CXCR6. ESRD was also associated with a shift in MAIT PMA-induced cytokine production away from IFNγ production and toward granulocyte macrophage-colony stimulating factor (GM-CSF) secretion, and a loss of E. coli-stimulated tumor necrosis factor α expression. Loss of IFNγ expression was associated with a combination of age, alterations in Tbet and Eomes expression, and inflammatory plasma cytokine levels. Conclusion: The loss of peripheral blood MAIT cells and associated shifts in tissue homing receptor expression and GM-CSF production may contribute to an immune environment that is permissive to bacterial replication, particularly in the lungs

Phenotypic and functional characterization of pharmacologically expanded Vγ9Vδ2 T cells in pigtail macaques

Summary: While gaining interest as treatment for cancer and infectious disease, the clinical efficacy of Vγ9Vδ2 T cell-based immunotherapeutics has to date been limited. An improved understanding of γδ T cell heterogeneity across lymphoid and non-lymphoid tissues, before and after pharmacological expansion, is required. Here, we describe the phenotype and tissue distribution of Vγ9Vδ2 T cells at steady state and following in vivo pharmacological expansion in pigtail macaques. Intravenous phosphoantigen administration with subcutaneous rhIL-2 drove robust expansion of Vγ9Vδ2 T cells in blood and pulmonary mucosa, while expansion was confined to the pulmonary mucosa following intratracheal antigen administration. Peripheral blood Vγ9Vδ2 T cell expansion was polyclonal, and associated with a significant loss of CCR6 expression due to IL-2-mediated receptor downregulation. Overall, we show the tissue distribution and phenotype of in vivo pharmacologically expanded Vγ9Vδ2 T cells can be altered based on the antigen administration route, with implications for tissue trafficking and the clinical efficacy of Vγ9Vδ2 T cell immunotherapeutics

SARS‐CoV‐2‐specific CD8 +

In‐depth understanding of human T cell‐mediated immunity in COVID‐19 is needed if we are to optimize vaccine strategies and immunotherapies. Identification of SARS‐CoV‐2 T cell epitopes and generation of peptide‐HLA tetramers facilitates direct ex vivo analyses of SARS‐CoV‐2‐specific T cells and their T cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS‐CoV‐2 epitopes restricted by HLA‐A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike‐derived peptides generated CD8(+)IFN‐γ(+) responses above background, S(1208‐1216) (QYIKWPWYI), S(448‐456) (NYNYLYRLF) and S(193‐201) (VFKNIDGYF), with S(1208) generating immunodominant CD8(+)IFN‐γ(+) responses. Using peptide‐HLA‐I tetramers, we performed direct ex vivo tetramer enrichment for HLA‐A*24:02‐restricted CD8(+) T cells in COVID‐19 patients and pre‐pandemic controls. The precursor frequencies for HLA‐A*24:02‐restricted epitopes were within the range previously observed for other SARS‐CoV‐2 epitopes for both COVID‐19 patients and pre‐pandemic individuals. Naïve A24/SARS‐CoV‐2‐specific CD8(+) T cells increased ~7.5‐fold above the average precursor frequency during COVID‐19, gaining effector and memory phenotypes. Ex vivo single‐cell analyses of TCRαβ repertoires found that the A24/S(448) (+)CD8(+) T cell TCRαβ repertoire was driven by a common TCRβ chain motif, while the A24/S(1208) (+)CD8(+) TCRαβ repertoire was diverse across COVID‐19 patients. Our study provides an in depth characterisation and important insights into SARS‐CoV‐2‐specific CD8(+) T cell responses associated with a prominent HLA‐A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID‐19 and could be exploited in vaccine or immunotherapeutic approaches

Anti-influenza hyperimmune immunoglobulin enhances Fc-functional antibody immunity during human influenza infection

Background: New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralizing antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. Methods: We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fcγ receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Results: Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1–3 days against infecting and noninfecting strains of influenza. Conclusions: Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus, and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralizing antibodies in the Flu-IVIG preparation

image_1_Mucosal-Associated Invariant T Cells Are Depleted and Exhibit Altered Chemokine Receptor Expression and Elevated Granulocyte Macrophage-Colony Stimulating Factor Production During End-Stage Renal Disease.PDF

Background<p>End-stage renal disease (ESRD) is associated with an increased susceptibility to infectious diseases, including infection with Mycobacterium tuberculosis (Mtb). Mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites produced by many bacterial species, including Mtb, and may play an important role in providing protective immunity against tuberculosis infection in the lung. To date, little is known about MAIT cell frequency, phenotype, or function in ESRD patients.</p>Methods<p>MAIT cells, identified by surface marker expression or MR1 tetramer binding, were characterized in 20 ESRD and 20 healthy control participants by multicolor flow cytometry. Ex vivo MAIT cell phenotype and cytokine production following PMA/ionomycin, IL-12/IL-18, or Escherichia coli stimulation were determined. Monocyte phenotype and plasma C-reactive protein/inflammatory cytokine levels were quantified by flow cytometry, ELISA, and multiplex bead array.</p>Results<p>Peripheral blood MAIT cells were significantly depleted among ESRD patients compared to controls by both phenotypic and tetramer analysis and exhibited a loss of CXCR3 expression coupled to increased expression of CCR6 and CXCR6. ESRD was also associated with a shift in MAIT PMA-induced cytokine production away from IFNγ production and toward granulocyte macrophage-colony stimulating factor (GM-CSF) secretion, and a loss of E. coli-stimulated tumor necrosis factor α expression. Loss of IFNγ expression was associated with a combination of age, alterations in Tbet and Eomes expression, and inflammatory plasma cytokine levels.</p>Conclusion<p>The loss of peripheral blood MAIT cells and associated shifts in tissue homing receptor expression and GM-CSF production may contribute to an immune environment that is permissive to bacterial replication, particularly in the lungs.</p