Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Cortical movement of Bicoid in early <i>Drosophila</i> embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model

<div><p>The Bicoid (Bcd) protein gradient in <i>Drosophila</i> serves as a paradigm for gradient formation in textbooks. The SDD model (synthesis, diffusion, degradation) was proposed to explain the formation of the gradient. The SDD model states that the <i>bcd</i> mRNA is located at the anterior pole of the embryo at all times and serves a source for translation of the Bicoid protein, coupled with diffusion and uniform degradation throughout the embryo. Recently, the ARTS model (active RNA transport, synthesis) challenged the SDD model. In this model, the mRNA is transported at the cortex along microtubules to form a mRNA gradient which serves as template for the production of Bcd, hence little Bcd movement is involved. To test the validity of the SDD model, we developed a sensitive assay to monitor the movement of Bcd during early nuclear cycles. We observed that Bcd moved along the cortex and not in a broad front towards the posterior as the SDD model would have predicted. We subjected embryos to hypoxia where the mRNA remained strictly located at the tip at all times, while the protein was allowed to move freely, thus conforming to an ideal experimental setup to test the SDD model. Unexpectedly, Bcd still moved along the cortex. Moreover, cortical Bcd movement was sparse, even under longer hypoxic conditions. Hypoxic embryos treated with drugs compromising microtubule and actin function affected Bcd cortical movement and stability. Vinblastine treatment allowed the simulation of an ideal SDD model whereby the protein moved throughout the embryo in a broad front. In unfertilized embryos, the Bcd protein followed the mRNA which itself was transported into the interior of the embryo utilizing a hitherto undiscovered microtubular network. Our data suggest that the Bcd gradient formation is probably more complex than previously anticipated.</p></div

αTubulin 67C and Ncd Are Essential for Establishing a Cortical Microtubular Network and Formation of the Bicoid mRNA Gradient in Drosophila.

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localized at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfil the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. We report hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (i) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (ii) long MTs up to 50 µm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the bcd mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. Our data strongly corroborate the ARTS model and explain the occurrence of the bcd mRNA gradient

Correction: Cortical movement of Bicoid in early <i>Drosophila</i> embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion model

Correction: Cortical movement of Bicoid in early <i>Drosophila</i> embryos is actin- and microtubule-dependent and disagrees with the SDD diffusion mode

In unfertilized embryos, <i>bcd</i> mRNA and Bcd protein move to the interior.

<p>Unfertilized eggs collected from different time intervals, 0–1 h (A-D), 1–2 h (E-H), 2–3 h (I-L), 3–4 h (M-P), stained for Bcd protein (A, E, I, M), Staufen protein (B, F, J, N) and DAPI (C, G, K, O). The merge of all staining patterns is revealed in (D, H, L, P). Note the weak diffusion of the Bcd protein away from the bulk (A, E, I, M), which is always congruent to the Staufen protein (B, F, J, N). (Q-T<i>) in situ</i> hybridization of <i>bcd</i> mRNA of 0–1 h (Q), 1–2 h (R), 2–3 h (S) and 3–4 h (T) old unfertilized embryos. Arrows in (Q, R, S) denote fast movement of a portion of mRNA particles, apparently not associated with Staufen (compare to B, F J). Time intervals and proteins are indicated in yellow.</p

Cortical movement of Bcd in hypoxic embryos and effects of hypoxia on the segmental axis.

<p>Pictures represent midsagittal confocal planes of embryos oriented with their dorsal side up and anterior to the left, except for (M-P) which show cuticles. Relative intensities of the crude confocal pictures were converted to a color scale with values of 0–255 (8-bit), shown in (D), except for (M) to (Q) and (S). Nomenclature of nuclear cycles follows that of [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185443#pone.0185443.ref046" target="_blank">46</a>]. (A-D) untreated <i>bcd</i>^<i>+5+8</i> embryos stained with Bcd antibodies. (A) interphase of nuclear cycle (nc) 6, (B) nc 7, (C) nc 8, (D) nc 9 embryos. Note the migration of the protein at the cortex of the embryo and not to the interior. (E-I) Relative Bcd intensities of nc 6 embryos in hypoxic <i>bcd</i>^<i>+5+8</i> embryos and collected at different time intervals after hypoxia treatment: (E) 1–2 h, (F) 2–3 h, (G) 3–4 h, (H) 7–8 h, (I) 17–18 h. Note the movement of the Bcd protein in the “sleeping” embryos along the cortex. (J) Formaldehyde-fixed nc 6 embryo stained with Bcd antibodies. (K-L) distribution of the <i>bcd</i> RNA in <i>bcd</i>^<i>+5+8</i> embryos after 2–3 h hypoxia (K) and 3–4 h hypoxia (L). Note the sparse movement of the mRNA. Weak (M) and strong cuticle phenotype (N) of wild-type (Oregon R) embryos collected in a 1 hour interval, subjected to 3 h hypoxia and recovered for 36 h. Weak (O) and strong cuticle phenotype (P) of <i>bcd</i>^<i>+5+8</i> embryos. (Q) 0–1 h collected, 3 h hypoxia-treated and 3 h recovery nc 14 embryo, stained for Bcd protein (green) and Eve (red), percentages indicate position of Eve stripes 1 and 7 in % egg length, respectively. Insert in (Q) shows DAPI staining of the posterior end demonstrating lack of pole cells. (R) color conversion of the Bcd pattern in (Q). (S) nc 14 <i>bcd</i>^<i>+5+8</i> embryo stained for Bcd protein (green) and Eve (red). Percentages indicate position of Eve stripes 1 and 7 in % egg length, respectively. (T) color conversion of the Bcd pattern in (S).</p

Bcd movement and stability in hypoxia-treated embryos depends on actin.

<p>Pictures represent midsagittal confocal planes of embryos oriented with their dorsal side up and anterior to the left. Relative intensities of the crude confocal pictures were converted to a color scale with values of 0–255 (8-bit), shown in (D). (A-C) nc 6 <i>bcd</i>^<i>+5+8</i> embryos treated with latrunculin B for 1–2 h (A), 2–3 h (B), 3–4 h (C) and stained for Bcd. (D) nc 5 embryo treated for 2–3 h with latrunculin B and stained for the <i>bcd</i> mRNA. Note that the Bcd protein does not move anymore along the cortex. (E-G) nc 6 <i>bcd</i>^<i>+5+8</i> embryos treated with phalloidin for 1–2 h (E), 2–3 h (F) and 3–4 h (G) and stained for Bcd. (H) nc 5 embryo treated for 2–3 h with phalloidin and stained for the <i>bcd</i> mRNA. Arrows in (E-G) denote interior nuclei, as well as energids revealing accumulation of Bcd protein. Note the decreased stability of Bcd in latrunculin B-treated embryos (A-C).</p

Independence of the early MT network from the actin sheet.

<p>(A)–(C) mid-sagittal confocal sections of the anterior tip of a nc 2 embryo stained with Phalloidin (A) to reveal the actin structure, with mab YL_1,2 against tyrosinated Tubulin (B) and merge in (C). (D)–(F) mid-sagittal section of a ventral region about 50 µm away from the anterior tip of a nc 6 embryo, stained with Phalloidin (D), mab YL_1,2 (E) and merge in (F). (G) 3-D reconstruction of the confocal stack of the embryo of (A)–(C), view is from the middle (M) to the more lateral (L) part of the embryo. For film of 3D view, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112053#pone.0112053.s005" target="_blank">Video S2</a>. (H) 3-D reconstruction of the confocal stack of the embryo of (D)–(F), view is identical as in G. For film of 3D view, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112053#pone.0112053.s006" target="_blank">Video S3</a>. The red background on the inner “roof” of the embryos in (G) and (H) is excess of free tubulin which could not be removed during background subtraction of the 3D-program. Stages of embryos are denoted in yellow and follow the nomenclature of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112053#pone.0112053-Foe2" target="_blank">[61]</a>.</p

Behaviour of the MT threads upon treatment with drugs: use of Taxol results in artefacts.

<p>Anterior (A, C, E) and posterior (B, D, F) ends of embryos treated with Taxol (A–D) or Colchicine & Colcemide (E, F) stained with mab YL_1,2 to reveal the MT network. (A, B) unfertilized embryo, (C, D) nc 5 embryo, (E, F) nc 6 embryo. Note that Taxol induces the formation of threads even in unfertilized embryos (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112053#pone-0112053-g001" target="_blank">Fig. Fig. 1D, E</a>) and even in posterior halves (B, D). Stages of embryos are denoted in yellow and follow the nomenclature of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112053#pone.0112053-Foe2" target="_blank">[61]</a>.</p