Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Panel (a) of this figure depicts the expression of the T cell activation marker GITR on CD25+, CD25 bright and CD25 verybright cells from healthy controls (white bars, n = 24) and SSc patients having limited cutaneous SSc (light gray bars, n = 18), late diffuse SSc (dark gray bars, n = 22) and early diffuse SSc (black bars, n = 22) patients. In panel (b) the expression of CD62L on Tregs is investigated. CD25+ and CD25 bright cells from SSc and healthy controls express similar levels of CD62L, whereas CD25 verybright from SSc patient subsets exhibit lower levels of CD62L compared to those from healthy controls. Panel (c) reflects the expression of CD69 on Tregs from healthy donors and SSc patients. CD25+, CD25 bright and CD25 verybright cells from SSc patients express significant lower levels of CD69 than those from healthy donors. CD69 expression on CD25 bright and CD25 verybright cells from edSSc patients was significantly lower then that from ldSSc patients, and ldSSc expressed CD69 significantly lower than those from lSSc. In panel (d) the expression on CD3+ cells is shown for all investigated groups. In contrast with that observed on Tregs from SSc patients, CD69 expression on CD4+ cells was significantly higher in all SSc patient groups. Panel (e) reflects the potential association between CD69 expression on Tregs and disease duration. CD69 expression on T regs from patients with lSSc correlated with disease duration, whereas this was not the case either with ldSSc nor edSSc. In all figures the white bars represent healthy controls, whereas lSSc, ldSSc and edSSc patients are represented by light gray, dark gray and black bars, respectively.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Expressing, Activation Assay, Marker

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: Unsorted CD3+ (MACS bead isolated) were stimulated with PHA (5 µg/ml) and consecutively incubated with CD25 high CD127 - or CD25 low CD127 high cells for 5 days. Thereafter, CD3+ cells were incubated with 3 H -thymidine for 24 more hours after which 3 H -thymidine incorporation was measured. Panel (a) reflects the suppressive capacity of Tregs from healthy donors and SSc patients. Proliferation of CD3+ effector cells was effectively inhibited by T regulatory cells from healthy controls, whereas a clearly diminished suppressive activity was observed in the experiments with Tregs from SSc patients. Suppressive effect of Treg (CD25 high CD127 - ) and non-Tregs (CD25 low CD127 high ) is presented in black and white bars, respectively. Results are the mean and SEM of 6 separate experiments using cells from healthy donors (n = 9), lSSc (n = 7), ldSSc (n = 9) and edSSc (n = 7). Panel (b) represents the correlation of CD69 expression and Treg suppressive capacity in Tregs from the various groups under investigation. The percentage of CD69 positive regulatory T cells (CD25 high CD127 - ) correlates well with the percentage of inhibition of CD3+ cells in healthy controls (triangles), lSSc (diamonds), ldSSc (circles) and edSSc (squares). Panel (c) reflects the expression of intracellular TGFβ in Tregs from healthy controls and SSc patients as measured using intracellular flow cytometry. CD25 high CD127 - cells from all SSc patients express lower TGFβ levels compared to controls. Left panel reflects an representative individual from each group whereas the right panel displays the mean of each group comprising 6 individuals (per group) coming forth from 4 independent experiments.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Isolation, Incubation, Activity Assay, Expressing, Inhibition, Flow Cytometry

Journal: PLoS ONE

Article Title: Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

doi: 10.1371/journal.pone.0005981

Figure Lengend Snippet: (a) During the co-cultures of unsorted CD3+ cells with either Tregs (CD25 high CD127 - ) or non-Tregs (CD25 low CD127 high ) 10 or 25% plasma from an edSSc patient or healthy control was added to the culture. The graph represents data from 3 independent experiments using 3 healthy control cells, and plasma derived from two edSSc patients and two control individuals. (b) The effect of SSc plasma was evaluated by adding 10% to CD3+ cells for 24 hrs stimulated with PHA or unstimulated. As a control, CD69 expression was measured on CD3+ cells stimulated with PHA only. CD4 and CD25 high /FoxP3 high cells were gated based on the expression of these markers using flow cytometry. (c) CD69 expression and induction upon PHA mediated stimulation of CD4+ and CD25 high /FoxP3 high obtained from healthy donors, lSSc, ldSSc and edSSc patients was investigated using flow cytometry. One representative patient from each group is shown.

Article Snippet: For immunostaining and analysis by fluorescence-activated cell sorting (FACS), we used phycoerythrin (PE), allophycocyanin (APC) and fluorescein isothiocynate (FITC) conjugated mouse monoclonal antibodies (mAb) against human CD4, CD8, CD25, CD69, GITR (Miltenyi Biotec Inc., CA, USA), CD127 (eBioscience, CA, USA), CD62 (BD Bioscience, NJ, USA).

Techniques: Clinical Proteomics, Control, Derivative Assay, Expressing, Flow Cytometry

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Memory CD8 + T cells with a resident phenotype are present in murine BM after acute systemic infection. (A–C) Spleen and BM were analyzed for virus‐specific CD8 T cells, 172 days after systemic infection with LCMV Armstrong. Data are shown for one representative experiment with n = 4 mice, out of three independent experiments; (A) Frequency of LCMV GP 33‐41 ‐specific and NP 396‐404 ‐specific CD8 + T cells of all CD44 + CD8 + T cells (average + SD); (B) Representative FACS staining for CD69 and CD62L on LCMV GP 33‐41 ‐specific CD44 + CD8 + T cells from spleen and BM; (C) Paired analysis for the percentage of CD62L − CD69 + cells of all LCMV GP 33‐41 ‐specific or NP 396‐404 ‐specific memory CD8 + T cells in spleen versus BM. (D–F) Analysis of OVA‐specific CD8 T cells in spleen and BM of WT mice, 49 days after systemic infection with Listeria‐OVA. Data are shown for one representative experiment with n = 9 mice, out of two independent experiments; (D) Frequency of OVA 257‐264 ‐specific CD8 + T cells of all CD44 + CD8 + T cells (average + SD); (E) Representative FACS staining for CD69 and CD62L on OVA 257‐264 ‐specific CD44 + CD8 + T cells from spleen and BM; (F) Paired analysis for the percentage of CD62L + CD69 + cells of all OVA 257‐264 ‐specific memory CD8 + T cells in spleen vs BM. (G–I) Expression levels of (G) CXCR3, (H) CXCR6, and (I) CX 3 CR1 in D b ‐GP 33‐41 ‐specific memory CD8 + T cell subsets in BM, 60 days after systemic infection with LCMV Armstrong. Data are shown for one representative experiment with n = 10 mice, out of two independent experiments. Data were analyzed by two‐tailed t ‐test, with matching (D, F), one‐way ANOVA followed by Tukey's multiple comparisons test (G–I) and two‐way ANOVA, with matching, followed by Bonferroni's multiple comparison test (C). Significance is indicated by * p < 0.05; ** p < 0.01; and **** p <0.0001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Virus, Staining, Expressing, Two Tailed Test, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Generation of BM CD8 + T RM cells does not require local infection or antigen presentation. (A) Percentage of CD69 + CD62L − cells within Influenza NP 366‐374 ‐specific CD44 + CD8 + T cells in spleen and BM 33 days after intranasal infection with Influenza A/HKx31. Data are shown from one representative experiment with n = 5 mice, out of two independent experiments; (B) Naïve WT mice were epicutaneously infected with HSV‐1 one day after i.v. transfer of 5 × 10 4 gBT‐I CD8 + T cells, which recognize the HSV‐1 K b ‐gB 498‐505 epitope and are identified as Vα2 + Thy1.1 + . Expression of CD69 was analyzed on donor cells in spleen and BM 40 days after infection; (C) Percentage of CD69 + within gBT‐I CD8 + T cells in spleen and BM, 11 months after in vitro activation and transfer; for (B) and (C), experiments were performed twice. (D) Frequency of gBT‐I cells within total CD8 α + cells in the spleen (left) or CD69 + within gBT‐I cells in the BM (right) of conjoined mice that received gBT‐I and were subsequently infected with HSV, as shown in (B); (E) Frequency of gBT‐I cells within total CD8 α + cells in the spleen (left) or CD69 + within gBT‐I cells in the BM (right) of conjoined mice that received in vitro activated gBT‐I cells, as shown in (C). For (D) and (E), organs were analyzed 3 weeks after conjoining. For (D) and (E), data are shown from one experiment with n = 3 mice. Data were analyzed by two‐tailed t ‐test, with matching. Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Immunopeptidomics, Expressing, In Vitro, Activation Assay, Two Tailed Test

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Virus‐specific resident‐like CD8 + T cells are found within human BM. (A) Representative staining for CD69 and CD103 on CD8 + CD3 + T cells in human BM; (B and C) Expression of chemokine receptors CXCR6, CCR5, and CX 3 CR1 on CD69 − and CD69 + CD8 + T cells in human BM; (B) Representative histograms for CD69 − and CD69 + CD8 + T cells; (C) Expression levels depicted as geoMFI for the same populations. For (A–C), data are shown for n = 4 non‐HLA typed BM samples from four different donors; (D) Combinatorial coding analysis of human CD8 + BM T cells specific for CMV or EBV. Dually labelled MHC‐I tetramers loaded with the same immunodominant peptide from either CMV‐VTE, CMV‐TPR, or EBV‐RAK identify virus‐specific CD8 + T cells from BM of HLA‐typed donor #1 (percentages of specific cells is indicated in the gate); (E–G) Quantification of CMV‐ or EBV‐specific CD8 T cells and the percentage of CD69 + cells therein, in human BM. Data are shown for n = 5 HLA‐typed BM samples from five different donors; (E) Table describes detailed information on each specific T cell population detected; (F) The percentage of Tet+ cells detected within total CD8 + cells. For (F) and (G), results are shown as average ± SD. In panel (G), only those samples are indicated for which >100 CD69 + tetramer + CD8 + T cells were acquired. Data was analyzed by one‐way ANOVA followed by Tukey's multiple comparisons test (G) and two‐way ANOVA, with matching, followed by Bonferroni's multiple comparison test (C). Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Virus, Staining, Expressing, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Murine steady‐state BM lodges CD8 + T cell memory‐like cells with a resident phenotype. (A–D) Analysis of the CD8 + T cell memory‐like compartment in adult mice under SPF breeding. Data are shown for 1 experiment with n = 5 mice; (A) Frequency of memory CD44 + cells within CD8 αβ + TCR β + T cells in peripheral blood (PB), spleen, and BM; (B) Frequency of CD69 + cells of all CD44 + CD8 αβ + TCR β + T cells; (C) Representative FACS plots showing expression of CD62L and CD69 on memory CD8 T cells (defined as CD44 + CD8 αβ + TCR β + cells) in the different organs; (D) Frequency of memory CD8 + T cells subsets in spleen and BM, as gated for in panel C; (E–H) Relative mRNA expression of (E) Ccr7 , (F) Klf2 , (G) S1pr1 , and (H) Hobit relative to the housekeeping gene Cyclophilin A in sort‐purified memory CD8 + T cell subsets from BM, as defined in (C) and (D). Data is shown for four independent sorting experiments, each with pooled BM from n = 4 mice; (I) Heatmap of RNA sequencing data for the 30 genes that compose the universal transcriptional signature of lymphocyte tissue residency in CD8 + T NV (CD44 − CD62L + ), T CM (CD44 + CD62L + ), T EM (CD44 + CD62L − CD69 − ), and T RM (CD44 + CD62L − CD69 + ) cells that were sort‐purified from BM of naïve mice. Data are shown from three independent sorting experiments, each with pooled BM from n = 6 mice. Relative expression levels ( Z ‐scores) of genes are shown, color coded according to legend. Data are shown as mean ± SD (A and B) or mean ± SEM (E–H). Statistical analysis was performed with one‐way ANOVA followed by Tukey's multiple comparisons test (A and B) and two‐way ANOVA followed by Bonferroni's multiple comparison test (D–H). Significance is indicated by * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Expressing, Purification, RNA Sequencing, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Specific features of BM CD8 + T RM cells. (A–D) Analysis of RNA sequencing data obtained for CD8 + T NV (CD44 − CD62L + ), T CM (CD44 + CD62L + ), T EM (CD44 + CD62L − CD69 − ), and T RM (CD44 + CD62L − CD69 + ) cells that were sort‐purified from BM of naïve mice. Data are shown from three independent sorting experiments, each with pooled BM from n = 6 mice; (A) Number of differentially expressed (DE) genes (downregulated in white, upregulated in black) between T RM versus T EM , T RM versus T CM , and T RM versus T NV . DE expressed genes were specifically up‐ or downregulated in BM T RM in comparison to their circulating counterparts, as determined by RNA sequencing (fold change [FC] > 1.5; and reads per kilobase per million mapped reads (RPKM) > 8]; (B) Venn diagram showing the overlap between genes DE by T RM and T EM , T RM , and T CM cells; (C) Heatmap displays relative amounts of transcripts of the 39 DE genes that were shared in the comparison shown in (B). Relative expression levels ( Z ‐scores) of genes are shown, color coded according to legend; (D) Correlation of gene ontology (GO)‐terms enriched in the sum of DE expressed genes between T RM versus T EM and T RM versus T CM (547 DE genes, FC > 1.5; RPKM = 8). GO‐terms were filtered to contain between 20 and 100 genes. Enrichment analysis revealed enrichment of the DE genes in 104 GO‐terms, which clustered into nine groups by comparison of their overlap index. Results are shown as a heatmap of the overlap indices, with the clustering and a color representation of each group on the left and a representative group name on the right; (E and F) Transcript levels of IFN‐γ in T CM , T EM , and T RM sorted from steady‐state mice, analyzed via RNAseq (E, obtained as in A–D) or RT‐qPCR (F, data are from four different sorting experiments, each with pooled BM from n = 4 mice); (G–I) Production of IFN‐γ by T CM , T EM , or T RM CD8 + T cells from LCMV‐infected mice. BM cells were incubated for 5 h with Brefeldin A in the absence (no stim.) or presence of GP 33‐41 peptide and IFN‐γ production was evaluated by ICS in the different memory populations. Data are shown for one representative experiment with n = 4 mice, out of two independent experiments; (G) Representative plots and quantification of the production of IFN‐γ in the absence (H) or presence of LCMV peptide (I); (J and K) Polyfunctionality of T EM and T RM cells was analyzed as in panels (G–I). Data are from one representative experiment with n = 5 mice, out of two independent experiments; (J) Co‐production of IFN‐γ and CCL3 by T EM or T RM CD8 + T cells from LCMV‐infected mice. Representative plots are shown; (K and L) Representative plots (K) and quantification (L) of T EM and T RM capable of simultaneously producing two, three, or four out of four cytokines (IFN‐γ, CCL3, IL‐2, and TNF‐α). Results in (H), (I), and (L) show mean ± SD. Statistical analysis was performed by one‐way ANOVA followed by Tukey's multiple comparisons test. Significance is indicated by ** p < 0.01 and *** p < 0.001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: RNA Sequencing, Purification, Comparison, Expressing, Quantitative RT-PCR, Infection, Incubation

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: BM CD8 + T RM cells reside in the parenchyma in close contact with the circulation. (A and B) Comparison of DE genes between T RM from different organs. RNA sequencing data for BM T RM was acquired as in Fig. I, while data for liver, SI, and skin T RM were obtained from ref. ; (A) Venn diagram showing the overlap between DE genes by T RM from BM, Liver, SI, and skin, compared to splenic T NV (Sp) (FC > 1.5; RPKM = 8); (B) Correlation plot depicting the similarity between T RM in different organs. The correlation is pairwise, and was calculated using log RPKM values of the 136 DE genes that were significant in all four contrasts; (C–E) Intravascular staining to probe the localization of T RM within the BM. Mice were i.v. injected with 3 μg of an antibody against CD8 a . Organs were obtained 2 min after injection and processed immediately. Data are shown for one representative experiment with n = 3 mice, out of two independent experiments; (C) A representative histogram comparing the staining with the anti‐CD8 α antibody in CD8 β + CD44 + cells from spleen, peripheral blood (PB), liver, and BM; geoMFI of CD8 α staining in (D) CD8 β + CD44 + cells from the different organs or (E) CD8 β + memory subsets within the BM; (F and G) Immunofluorescence analysis of (F) CD8 α + cells or (G) CD69 + CD8 α + cells within steady‐state BM. Scale bar = 50 μm. Vasculature was visualized with antibodies against CD31 and CD144 and nuclei with Helix NP green staining. One representative region from a tile scan of 4 × 7 images is shown. The full tile scan is shown in Supporting Information Fig. ; 40× magnification. Each CD8 a + cell is encircled in white to identify these cells across the different stainings. CD69 + CD8 α + cells are marked with an arrow.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Comparison, RNA Sequencing, Staining, Injection, Immunofluorescence

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Maintenance of BM CD8 + T RM cells depends on IL‐15 and Hobit. (A–C) In vitro‐activated gBT‐I cells were transferred into WT or IL‐15 −/− mice and, after 30 days, the presence of total gBT‐I and CD69 + gBT‐I was analyzed in the BM. Results are shown as (A) representative plots, (B) quantification of the % of gBT‐I within total CD8 α + cells and (C) quantification of TRM (CD69 + ) cells within gBT‐I cells in the BM, from one experiment with n = 5 WT and 4 IL‐15 −/− mice. Results show mean ± SD; experiment was performed twice. (D and E) WT (Blimp +/+ Hobit +/+ ) or mice deficient for Hobit (Blimp +/+ Hobit −/− ), Blimp1 (Blimp −/‐ Hobit +/+ ), or both (Blimp −/− Hobit −/− ) were infected i.n. with Influenza A/HKx31. BM was harvested and analyzed 63 dpi. Results are shown from one representative experiment with n = 5 mice, out of three independent experiments, as (D) percentage of D b ‐NP 366‐374 + cells within total CD8 αβ + T cells and (E) percentage of T RM (CD69 + CD62L − ) cells within D b ‐NP 366‐374 + CD8 αβ + T cells. Data were analyzed by two‐tailed t ‐test (B and C) or one‐way ANOVA followed by Tukey's multiple comparisons test (D and E). Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: In Vitro, Infection, Two Tailed Test

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: The pool of BM CD8 + T RM cells is expandable. (A–C) Distribution of CD8 αβ + memory populations specific for mCMV acute and inflationary epitopes analyzed 65 days p.i.; Representative plots are shown for (A) acute epitopes M45 and M57 and (B) inflationary epitopes M38 and m139. Tetramer stainings are shown in the left panels and the gating for T RM (CD62L − CD69 + ) and T EM (CD62L − CD69 − ) is shown in the right panels; (C) Percentage of T EM and T RM within live BM cells for each mCMV‐specific population. Data is shown for one representative experiment with n = 4 mice, out of two independent experiments; (D–F) Distribution of OT‐I memory populations analyzed more than 30 days after the last immunization in mice that received one or three homologous boosts to generate OVA‐specific memory. Data are shown for one experiment with n = 5 mice that received one challenge and 4 mice that received three challenges; (D) Schematic depiction of the experimental setup; (E) Percentage of OT‐I cells within total CD8 α + cells in the BM; (F) Percentage of T CM , T EM , and T RM within OT‐I cells in the BM after one or three homologous challenges; (G–I) Distribution of OVA‐specific CD8 αβ + memory populations analyzed more than 60 days after the first challenge in mice that received only Lm‐OVA or Lm‐OVA followed by LCMV Armstrong infection, more than 30 days after the first challenge. Data are shown for one experiment with n = 5 mice/group. Results from the reverse experiment are shown in Supporting Information Fig. ; (G) Schematic depiction of the experimental setup; (H) Percentage of K b ‐OVA 257‐264 + T RM cells of live BM cells; (I) Percentage of total T RM within live BM cells. Data were analyzed by two‐tailed t ‐test (E, H, and I) and two‐way ANOVA, followed by Bonferroni's multiple comparison test (C and F). Significance is indicated by ** p < 0.01 and *** p < 0.001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Two Tailed Test, Comparison

Journal: Immunity

Article Title: Low-Avidity CD4 + T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19

doi: 10.1016/j.immuni.2020.11.016

Figure Lengend Snippet:

Article Snippet: CD69-PE (REA824) , Miltenyi Biotec , Cat#130-112-613; RRID: AB_2659065.

Techniques: Functional Assay, Recombinant, Staining, Sequencing, Gene Expression, Software

Journal:

Article Title: c-Myb regulates lineage choice in developing thymocytes via its target gene Gata3

doi: 10.1038/sj.emboj.7601801

Figure Lengend Snippet: Phenotypes of loss or gain of c-Myb during positive selection coincide with high levels of c-Myb expression. (A) Percentage distribution of thymocyte subsets measured by expression of CD8 (x-axis) and CD4 (y-axis) in: a MybF/F littermate control versus a MybF/F:CD4Cre mouse (top panels); a MybF/FOTIIRAG2−/− control versus a MybF/F:CD4Cre OTIIRAG2−/− mouse (centre panels), and an age-matched normal control versus a vMyb4 transgenic (lower panels). Data are representative plots. (B) Dot plot showing expression of CD69 (x-axis) and TCRβ (y-axis) on thymocyte subpopulations used in this study. Subsets boxed as (A–D) are successively more mature (C) q-RT–PCR quantitating c-Myb mRNA levels relative to hprt in thymocyte subsets (A–D) from MHC Class I (black bars; CD4 only) and MHC Class II (white bars; CD8 only) mice. Data are the mean of ⩾5 separate experiments. Error bars: standard deviation (s.d.).

Article Snippet: Purified thymocyte populations were prepared using anti-CD69 biotinylated antibody and streptavidin-conjugated magnetic beads (Miltenyi Biotec) as follows: both positively and negatively selected CD69 fractions were stained with anti-CD4, anti-CD8 and anti-CD69 antibodies and sorted to isolate DPCD69 + , CD4 + CD8 lo , CD4SP and CD8SP subpopulations from the CD69 + fraction.

Techniques: Selection, Expressing, Control, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal:

Article Title: c-Myb regulates lineage choice in developing thymocytes via its target gene Gata3

doi: 10.1038/sj.emboj.7601801

Figure Lengend Snippet: c-Myb and Gata3 expression following TCR signalling. (A) qRT–PCR quantitating c-Myb expression relative to hprt. mRNA was harvested from naïve DP thymocytes stimulated for the times shown on the x-axis with nothing (grey lines), or αCD3 (black lines). Data are the mean of three experiments performed in triplicate. Error bars: s.d. (B) qRT–PCR quantitating Gata3 mRNA relative to hprt mRNA from the same experiments. (C) Gata3 expression assessed by Western blot relative to a β-actin control. Cell lysates were prepared from purified DP CD69− thymocytes of the genotypes shown, stimulated in vitro with PMA and ionomycin for the indicated times. (D) c-Myb and Gata3 expression following αCD3 stimulation of MHCI/IIo/o DP thymocytes in the presence or absence of UO126. Data are the mean of three experiments performed in triplicate. Error bars: s.d.

Article Snippet: Purified thymocyte populations were prepared using anti-CD69 biotinylated antibody and streptavidin-conjugated magnetic beads (Miltenyi Biotec) as follows: both positively and negatively selected CD69 fractions were stained with anti-CD4, anti-CD8 and anti-CD69 antibodies and sorted to isolate DPCD69 + , CD4 + CD8 lo , CD4SP and CD8SP subpopulations from the CD69 + fraction.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Purification, In Vitro