Mycobacterium tuberculosis peptides presented by HLA-E molecules are targets for human CD8 T-cells with cytotoxic as well as regulatory activity

Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8(+) T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8(+) T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8(+) T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-beta. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8(+) T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity

The wide-field, multiplexed, spectroscopic facility WEAVE : survey design, overview, and simulated implementation

Funding for the WEAVE facility has been provided by UKRI STFC, the University of Oxford, NOVA, NWO, Instituto de Astrofísica de Canarias (IAC), the Isaac Newton Group partners (STFC, NWO, and Spain, led by the IAC), INAF, CNRS-INSU, the Observatoire de Paris, Région Île-de-France, CONCYT through INAOE, Konkoly Observatory (CSFK), Max-Planck-Institut für Astronomie (MPIA Heidelberg), Lund University, the Leibniz Institute for Astrophysics Potsdam (AIP), the Swedish Research Council, the European Commission, and the University of Pennsylvania.WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366-959 nm at R ∼ 5000, or two shorter ranges at R ∼ 20,000. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for ∼ 3 million stars and detailed abundances for ∼ 1.5 million brighter field and open-cluster stars; (ii) survey ∼ 0.4 million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey  ∼ 400 neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in z 1 million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at z > 2. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator.PostprintPeer reviewe

The wide-field, multiplexed, spectroscopic facility WEAVE: Survey design, overview, and simulated implementation

WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366$-$959\,nm at $R\sim5000$, or two shorter ranges at $R\sim20\,000$. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for $\sim$3 million stars and detailed abundances for $\sim1.5$ million brighter field and open-cluster stars; (ii) survey $\sim0.4$ million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey $\sim400$ neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in $z<0.5$ cluster galaxies; (vi) survey stellar populations and kinematics in $\sim25\,000$ field galaxies at $0.3\lesssim z \lesssim 0.7$; (vii) study the cosmic evolution of accretion and star formation using $>1$ million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at $z>2$. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator.Comment: 41 pages, 27 figures, accepted for publication by MNRA

The wide-field, multiplexed, spectroscopic facility WEAVE: Survey design, overview, and simulated implementation

WEAVE, the new wide-field, massively multiplexed spectroscopic survey facility for the William Herschel Telescope, will see first light in late 2022. WEAVE comprises a new 2-degree field-of-view prime-focus corrector system, a nearly 1000-multiplex fibre positioner, 20 individually deployable 'mini' integral field units (IFUs), and a single large IFU. These fibre systems feed a dual-beam spectrograph covering the wavelength range 366−959\,nm at R∼5000, or two shorter ranges at R∼20000. After summarising the design and implementation of WEAVE and its data systems, we present the organisation, science drivers and design of a five- to seven-year programme of eight individual surveys to: (i) study our Galaxy's origins by completing Gaia's phase-space information, providing metallicities to its limiting magnitude for ∼3 million stars and detailed abundances for ∼1.5 million brighter field and open-cluster stars; (ii) survey ∼0.4 million Galactic-plane OBA stars, young stellar objects and nearby gas to understand the evolution of young stars and their environments; (iii) perform an extensive spectral survey of white dwarfs; (iv) survey ∼400 neutral-hydrogen-selected galaxies with the IFUs; (v) study properties and kinematics of stellar populations and ionised gas in z1 million spectra of LOFAR-selected radio sources; (viii) trace structures using intergalactic/circumgalactic gas at z>2. Finally, we describe the WEAVE Operational Rehearsals using the WEAVE Simulator

Predicted HLA-E binding Mtb peptides induce T-cell proliferation.

<p>CFSE-labeled PBMCs of 20 donors were stimulated with 10 µg/ml of peptide in the presence of IL-7, on day 7 low dose IL-2 was added and at day 10 proliferation was assessed by flowcytometry. Plots are gated on CD3⁺CD8⁺CD56⁻ cells in the lymphocyte gate, the percentage of proliferation indicated is within CD3⁺CD8⁺CD56⁻ cells. Peptide and donor numbers correspond to the numbers in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000782#ppat-1000782-t001" target="_blank">Table 1</a>.</p

HLA-E binding peptide specific T-cell lines have immuno-regulatory activity.

<p>(A) Peptide specific T-cell lines were generated by stimulation with peptide in the presence of rIL-7 and further expanded with rIL-2. To investigate their potential capacity to inhibit CD4⁺ T-cell activity, they were co-cultured with a well characterized CD4 Th1 clone (Rp15 1-1 (1×10e4 cells per well)) proliferating to its cognate peptide (0.5 µg/ml) presented by HLA-DR3⁺ cells. Dose-dependent addition of peptide stimulated cells (ranging from 0.6–5×10e4 added T-cells) reduced proliferation of the Th1 clone as measured by [³H] TdR incorporation at day 3. T-cell lines generated from 3 donors directed against 3 different peptides are shown (donor 2 against peptide 62, donor 4 against peptide 55 and donor 6 against peptide 68), the T cell line from donor 2 against peptide 54 had a similar suppressive capacity (not shown) CPM = counts per minute. (B) To exclude that suppression was merely the consequence of lysis of the responder T-cells, we analysed cell survival in a CFSE labeling experiment. The responder T-cell clone Rp15 1-1 was labeled with a low dose of CFSE (0.005 µM), whereas a second, isogenic T-cell clone with a different peptide specificity and HLA-DR2 restriction (R2F10), was labeled with a high concentration of CFSE (5 µM). Both responder and irrelevant T-cell clones were HLA-E negative. They were then co-cultured with the HLA-E binding Mtb peptide specific T-cell lines (“Treg”) in the presence of the peptide (0.5 µg/ml) recognized by the responder clone Rp15 1-1 and HLA-DR3⁺ APCs. After 16 hours CFSE intensity was measured by flowcytometry. In the absence of added Tregs, similar numbers of responder and irrelevant T-cell clones were retrieved (ratio of 1). The addition of “Tregs” to peptide activated or control cultures also resulted in similar numbers of both responder and irrelevant T-cells, thus indicating that the responder clone is not lysed by the Tregs. Simultaneously a 3 day co-culture was performed and analyzed by [³H] TdR uptake. This experimental set-up revealed inhibition of proliferation of the responder clone. Addition of different numbers of Tregs did inhibit proliferation in a suppression assay but not the ratio of responder over irrelevant T-cell numbers in a CFSE intensity assay. (C) Clonal populations were obtained by limiting dilution of the T-cell line of donor 2 against peptide 62, all derived from 0.1 cells/well cultures. Clones were co-cultured in different ratios to Rp15 1-1 as described in (a) and 3H TdR incorporation measured. Three out of the 5 tested clones inhibited proliferation of the indicator clone in a dose dependent manner. (D) K562 target cells selectively expressing HLA-E were loaded with peptide 62 (10 µg/ml) before ⁵¹Cr labeling, followed by 5 hour co-incubation with T-cell clones and determination of ⁵¹Cr release. Clone 4G10 strongly lysed peptide loaded target cells, whereas 3E11 and 3C1 had moderate lysing capacity.</p

PPD responders and BCG vaccinated infants recognize predicted HLA-E binding peptides derived from Mtb.

<p>(A) Summary of all proliferation data from 10 PPD responders and 10 PPD non-responding donors. Proliferation was calculated using the following formula: Geometric mean of undivided population minus geometric mean of all cells. Subsequently the percentage was calculated by: (delta geo mean of sample- delta geo mean of negative control)/ delta geo mean of maximal proliferation (PHA). Samples with 10% specific proliferation or more were considered positive (black boxes). Blue highlights identify the peptides that were selected for detailed analysis. Umbilical cord blood (UCB) (n = 5) cell proliferation was assessed by the percentage of proliferating CD3⁺CD8⁺CD56⁻ cells and proliferation >10% was considered positive. (B) PBMCs from BCG vaccinated infants (n = 12) were tested in the same way as the cells from PPD responders for 10 selected peptides. Proliferation of 5–10% is indicated in grey and proliferation >10% in black. (C) Comparison of PPD responders, non-responders & cord blood samples for peptide recognition, i.e. the number of peptides recognized per donor (n = 10 for adults, n = 5 for cord blood). Analyzed using Students T-test with a p<0.05 considered significant. (D) CD8⁺ T cells were purified from PBMCs by magnetic bead separation of donors 2,4 and 6 and proliferation (CFSE) in response to peptide pre-pulsed K562 cells with (right plots) or without (left plots) HLA-E was assessed. Plots are gated on CD8⁺CD56⁻ cells and CFSE dilution is plotted.</p

Predicted HLA-E binding Mtb peptide specific T-cell lines are cytotoxic.

<p>(A) HLA-E expression on K562 cell lines after peptide loading. Upper graph, untransfected K562 cells do not stain with anti-HLA-E (3D12) antibodies, whereas HLA-E transfected K562 cells expressing the natural HLA-E ligand (HLA-B7) or cells loaded with a Mtb derived HLA-E binding peptide (#55, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000782#ppat-1000782-t001" target="_blank">Table 1</a>) express similar levels of HLA-E. Middle graph, similar staining is observed when an antibody against HLA class I (W6/32) is used, with equal expression levels of HLA-E in the presence of a natural ligand or an Mtb derived peptide. Bottom graph, the isotype control staining for 3D12 and W6/32 was negative on all cell lines tested. HLA-E expression was always confirmed before functional experiments. (B) Peptide specific T-cell lines were generated by stimulation with peptide in the presence of IL-7 and further expansion using IL-2. K562 cells expressing HLA-E were loaded with specific and control peptides, labeled with 1 µCi ⁵¹Cr and co-cultured with T-cell lines for 5 hours in different ratios before ⁵¹Cr release was measured. Data are expressed as % specific lysis. Left graph: T-cell line derived from donor 4 directed against peptide 55 (using the high affinity HLA-E binding peptide 68 or the natural B7 ligand as irrelevant control), right graph: T-cell line derived from donor 2 directed against peptide 62 (using peptide 68 on transfected K562, or peptide 62 on untransfected K562 as irrelevant controls). Representative of 4 lines derived from 3 donors. (C) Fully HLA-A,B,C mismatched adherent monocytes were infected with live BCG (MOI 5, overnight) before labeling with 1 µCi ⁵¹Cr. Cells were co-cultured with T-cell lines for 5 hours in 6-replicate cultures. Grey bars represent BCG infected monocytes whereas white bars represent uninfected monocytes. All HLA-E peptide restricted T-cell lines recognized BCG infected target cells. In contrast a CD8⁺ T-cell clone restricted to the male HY antigen presented in HLA-A2 did not lyse infected monocytes, whereas the natural ligand (B-cells expressing HY in the context of HLA-A2) was specifically lysed, resulting in up to 100% target cell lysis. Data are depicted as mean+standard error of the mean and represent multiple experiments.</p