Journal: Clinical & Translational Immunology

Article Title: Limited effect of duration of CMV infection on adaptive immunity and frailty: insights from a 27‐year‐long longitudinal study

doi: 10.1002/cti2.1193

Figure Lengend Snippet: CMV‐induced changes in the CD8 + T‐cell pool are established early after CMV infection. (a) Absolute numbers of CD8 + T‐cell subsets compared between CMV ‐ ( n = 113), ST CMV + ( n = 19), and LT CMV + individuals ( n = 136). (b) Relationship of duration of CMV infection with CD8 + T EMRA cells numbers. (c) CD8 + T EMRA cells numbers at study endpoint in individuals that seroconverted at younger age (≤ 38 years of age, n = 26) compared to those who converted recently at an older age (≥ 45 years of age, n = 18, mean age 58.5 ± 8.1). (d–g) Numbers and percentages of CMV‐specific CD8 + T cells ( d, e ), percentage of T EMRA cells of total CD8 + cells ( f ) and percentage of expression of KLRG‐1 (G) in HLA‐A2 positive individuals for pp65‐epitope NLVPMVATV compared between ST CMV + ( n = 8) and LT CMV + ( n = 4) individuals ( g ). (h, i) Percentages of IFNγ producing CD8 + T cells ( h ) and polyfunctional CD8 + T cells producing IFNγ, TNFα, Mip, CD107 and/or IL2 ( i ) after CMV‐specific peptide stimulation in ST CMV + and LT CMV + individuals ( n = 27). Boxplots show median with interquartile range. * P < 0.05, **** P < 0.0001.

Article Snippet: For the HLA‐A2 + individuals (12/20), tetramer staining was performed for 15 min at room temperature with CMV‐(A*0201/NLVPMVATV)‐APC (Immudex, Fairfax, VA, USA) after which extracellular staining was performed for 20 min at 4°C with Fixable Viability Staining‐780 (APC‐Cy7) (Thermo Fisher), CD3 APC‐R700(SK7)‐AF700(BD) CCR7(150503)‐BrilliantViolet(BV)395 (BD Biosciences), CD8 + (RPA‐T8)‐BV510, CD45RO+(UCHL1)‐BV711, CD27(O323)‐BV786, PD‐1(EH12.2H7)‐PerCP‐Cy5.5 (BioLegend) and KLRG‐1(13F12F2)‐PE‐Cy7 (Thermo Fisher).

Techniques: Infection, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: CD8+ T Cells Protect During Vein Graft Disease Development

doi: 10.3389/fcvm.2019.00077

Figure Lengend Snippet: T cell activation in vein grafts. (A) Blood, spleen, (non) draining lymph nodes, and vein grafts of C57BL/6 male mice were analyzed with FACS. CD44 and CD62L are used to quantify the percentage of effector T cells. Example of flow cytometry plot of a vein graft sample is shown. (B) The percentage of effector CD8+ T cells (CD44+ CD62L- of total CD8+ T cells) is shown. (C) The percentage of effector CD4+ T cells (CD44+ CD62L- of total CD4+ T cells) is shown. (D) T cell activation was analyzed with KLRG1 and CD62L. Example of flow cytometry plot of a vein graft sample is shown. (E) The percentage of activated CD8+ T cells (KLRG1+ CD62L- of total CD8+ T cells) is shown. (F) The percentage of CD4+ T cells (KLRG1+ CD62L- of total CD4+ T cells) is shown. (G) T cell activation was analyzed with CD25 and CD44. Example of flow cytometry plot of a vein graft sample is shown. (H) The percentage of activated CD8+ T cells (CD25+ CD44+ of total CD8+ T cells) is shown. (I) The percentage of CD4+ T cells (CD25+ CD44+ of total CD8+ T cells) is shown. (A–I) n = 4/group. Significant differences between blood, spleen, (non) draining lymph nodes compared to vein grafts are indicated. * P < 0.05, **** P < 0.0001, one-way ANOVA and Kruskal-Wallis test.

Article Snippet: Conjugated monoclonal antibodies to mouse CD11b (V450, eBioscience), Class II (V500, BD Horizon), Ly6C (fluorescein isothiocyanate [FITC], Biolegend), CD11c (phycoerythrin [PE], Biolegend), CD86 (Allophycocyanin [APC], Biolegend), F4/80 (PE-Cy7, Biolegend), Ly6G (Alexa Fluor 700, Biolegend), CD19 (APC-Cy7, eBioscience), CD44 (V450, eBioscience), CD8 (FITC, Biolegend), TCR Beta (PE, eBioscience), CD25 (APC, eBioscience), KLRG1 (PE-Cy7, eBioscience), CD62L (APC-Cy7, eBioscience), CD4 (Brilliant Violet 605, Biolegend) were used.

Techniques: Activation Assay, Flow Cytometry

Journal: Frontiers in Cardiovascular Medicine

Article Title: CD8+ T Cells Protect During Vein Graft Disease Development

doi: 10.3389/fcvm.2019.00077

Figure Lengend Snippet: TCR and co-stimulation independent VGD development. (A) Vein graft patency of control and OTI male mice is shown. Percentage of clearly pulsatile and occluded vein graft is shown in control and OTI mice, not statistically different ( p = 0.236). A 2-tailed Student's t -test was used. (B) The percentage of activated CD8+ T cells (CD8+ KLRG1+ CD62L-), and (C) effector CD8+ T cells (CD8+CD62L-CD44+) is shown of control and OTI mice in blood, spleen, (non) draining lymph nodes, and vein grafts. (A–C) control n = 4, OTI n = 5. Mann-Whitney test was used. (D) Production of inflammatory cytokines was measured in supernatant of DCs and VMSCs cultured with OTI T cells and Ovalbumin (Ova), stimulated with LPS for 24 h. As control groups, VSMCs were stimulated without OTI T cells or without Ovalbumin and OTI T cell with Ovalbumin were stimulated without VSMCs. The concentration of IFNγ was measured in supernatant. n = 6. ** P < 0.01, **** P < 0.0001. One-way ANOVA was used. (E) vein graft patency of control ( n = 11) and (F) CD70 −/− ( n = 12), (G) CD80/86 −/− ( n = 14), and (H) CD70/80/86 −/− ( n = 14) mice is shown 28 days after surgery. Percentage of clearly pulsatile, not clearly pulsatile and occluded vein graft is shown. A statistically significant difference was not shown ( p = 0.679), Kruskal-Wallis test was used.

Article Snippet: Conjugated monoclonal antibodies to mouse CD11b (V450, eBioscience), Class II (V500, BD Horizon), Ly6C (fluorescein isothiocyanate [FITC], Biolegend), CD11c (phycoerythrin [PE], Biolegend), CD86 (Allophycocyanin [APC], Biolegend), F4/80 (PE-Cy7, Biolegend), Ly6G (Alexa Fluor 700, Biolegend), CD19 (APC-Cy7, eBioscience), CD44 (V450, eBioscience), CD8 (FITC, Biolegend), TCR Beta (PE, eBioscience), CD25 (APC, eBioscience), KLRG1 (PE-Cy7, eBioscience), CD62L (APC-Cy7, eBioscience), CD4 (Brilliant Violet 605, Biolegend) were used.

Techniques: MANN-WHITNEY, Cell Culture, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Distinctive phenotype for HLA-E- versus HLA-A2-restricted memory CD8 αβT cells in the course of HCMV infection discloses features shared with NKG2C + CD57 + NK and δ2 - γδT cell subsets

doi: 10.3389/fimmu.2022.1063690

Figure Lengend Snippet: Immunophenotype of HLA-E UL40 and HLA-A2 pp65 memory CD8 T cells after a primary HCMV infection, a reactivation in KTR and during chronic infection in HCMV+ HV. After immunostaining and fluorescence acquisition, CD8Tcell populations were defined using a dedicated gating strategy as reported in the Materials and methods section and in Figure S3 in order to identify pMHC tetramer- CD8Tcells (tet-CD8T), HLA-E UL40 (E/UL40+) and HLA-A2 pp65 (A2/pp65+) CD8Tcells. Box plots with median and interquartile values showing the specific expression for Tim-3 (A) , Lag-3 (B) , 4-1BB (C) , 2B4 (D) , Tigit (E) and KLRG1 (F) . Results are expressed as percents of positive cells in the different CD8 T cell subsets. (A–F) Box plots with median and interquartile values for analyses performed on HLA-E UL40 CD8T cells from KTR with a primary infection (n=5), a reactivation (n=7), from HV with chronic infection/latency (HV; n=9) and on HLA-A2 pp65 CD8T cells from KTR with a primary infection (n=7), a reactivation (n=7), from HV with chronic infection/latency (n=10) and the respective tet - CD8T cells. Each point corresponds to an individual CD8T response. Statistical analysis was performed by Mann-Whitney U -test. P values: *for p < 0.05, **for p<0.01, ***for p<0.001 and ****for p<0.0001.

Article Snippet: Antibody included: BB700-anti-CD45RA (clone 5H9, BD Optibuild™, BD Biosciences), BV786-anti-CD45RO (clone UCHL1, BD Horizon™, BD Biosciences), PE-Cy7 anti-CCR7 (CD197, clone 3D12, BD Pharmingen™, BD Biosciences) BB515-anti-CD27 (clone M-T271, BD Horizon™), PE-anti-CD28 (clone CD28.2, BD Pharmingen™) and PE-anti-CD57 (clone NK1), BB700-anti-CD56 (clone B159), BB700-anti-HLA-DR (clone 46-6), BB515-anti-CD38 (clone HIT2), PE-anti-2B4 (CD244, clone 2-69), BV786-anti-4-1BB (CD137, clone 4B4-1), BB700-anti-PD-1 (CD279, clone EH12.1), PE-Cy7-anti-TIGIT (clone A15153G), BB515-anti-Tim-3 (CD366, clone 7D3), BB515-or FITC- anti-Lag-3 (CD223, clone T47-530), PE-Cy7-anti-KLRG1 (clone 2f1/KLRG1), BV786-anti-CX3CR1 (clone 2A9-1) and PE-anti-CD62L (clone DREG-56) all from BD Biosciences.

Techniques: Infection, Immunostaining, Fluorescence, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) CD8 + T mem response to influenza in mixed BM chimeras, generated as in . Mice were immunized with PR8 influenza, and the CD8 + T mem response of the CD45.2 donor to NP was assessed in lungs by tetramer on day 40. Quantitation shows frequency of (donor) CD45.2 + CD8 + T cells that are NP-specific (n = 5–6). **p < 0.01, by Mann–Whitney U-test (n = 4–7). ( B ) SLEC and MPEC populations in the Atg7 +/+ and Atg7 −/− antigen-specific CD8 + T cell pool. Mixed BM chimera generated as in , were immunized with MCMV 8 weeks after transplantation. Dot plots show example of KLRG1 and CD127 expression on gated CD45.2 + m45-tetramer + CD8 + T cells on day 10 post-infection. Upper bar graph depicts the % of CD45.2 + m45-tetramer + CD8 + T cells that are CD127 − KLRG1 + (SLECs). Lower bar graph shows the % of CD127 + KLRG1 − (MPECs) in the same population. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( C ) Markers of exhaustion on Atg7 −/− MCMV-specific CD8 + T cells on MCMV challenged BM chimera. Dot plots depict example of PD-1 and TIM-3 staining on gated CD45.2 + m45-tetramer + CD8 + T cells. Bar graph quantifies the percentage of (donor) CD45.2 + m45-tetramer + CD8 + T cells that are PD-1 + TIM-3 + at day 10 post-infection. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( D ) CD127 expression on Atg7 −/− MCMV-specific CD8 + T cells in MCMV challenged BM chimeras. Examples of CD127 staining on gated CD45.2 + m45-tetramer + CD8 + T cells from spleen on day 10 post-infection are shown. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( E ) IL-15Rα expression on splenic Atg7 −/− MCMV-specific CD8 + T cells in MCMV challenged BM chimeras. Histograms depict IL-15Rα expression in CD44 lo CD8 + T cells from unimmunized mice (grey dotted line), Atg7 +/+ CD45.2 + m45-tetramer CD8 + T cells (grey filled line), and Atg7 −/− CD45.2 + m45-tetramer CD8 + T cells (black line). The left histogram shows expression in control normal WT and T- Atg7 −/− mice, the right histogram indicates staining in donor CD45.2 + cells from BM chimera mice. Quantified is IL-15Rα mean fluorescence intensity on gated CD45.2 + m45-tetramer CD8 + T cells (n = 4–7). All values are mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.03706.008

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Generated, Quantitation Assay, MANN-WHITNEY, Transplantation Assay, Expressing, Infection, Staining, Control, Fluorescence

Journal: Cell reports

Article Title: PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8 + T cells at the expense of central memory

doi: 10.1016/j.celrep.2021.109804

Figure Lengend Snippet: (A–F) Viable CD8 + splenocytes from mice infected with LCMV Armstrong (n = 2, 4–5/group/time point). (A–C) NP396-specific CD8 + cells day 15 p.i. (A) CD127 and KLRG1 expression (left: representative staining; right: %KLRG1 + CD127 − ). (B) Representative histogram of CD27 (top) and CD127 (bottom). (C) GzmB and TCF1 staining. Middle: TCF1 MFI; right: GzmB MFI. (D–F) NP396-specific CD8 + cells day 58 p.i. (D) CD44 and CD62L staining. Representative flow (left), % CD44 hi CD62L lo cells (right). (E) CD127 histograms: CD44 hi CD62L lo (top) and CD44 hi CD62L + (bottom). (F) TCF1 MFI. (G) TCF1 staining of allo-reactive CD8 + cells from healthy controls and patients with APDS (n = 2, representative histogram). (H–L) Mice were infected with X31 and challenged with PR8 (n = 2, 3–5 mice/genotype/time point). (H) Infection outline. (I) PA224-specific CD8 + cell numbers. (J) IFN-γ and TNF-α from day 8 cells stimulated with either PA 224–233 (left) or anti-CD3 plus anti-CD28 (right). (K) NP366-specific CD8 + T cell numbers. (L) IFN-γ and TNF-α from day 35 cells stimulated with either NP 366–374 (left) or anti-CD3 plus anti-CD28 (right). (M–O) OT-1 cells were transferred into congenic hosts, infected with influenza X31-OVA and challenged with PR8-OVA (n = 2, 3 mice/genotype/time point). (M) Outline. (N) Viable OT-1 cell numbers. (O) TCF1 and GzmB expression in OT-1 cells on day 35. Representative experiment (n = 2). Graphs show mean ± SEM. *p < 0.05; **p < 0.01. See and .

Article Snippet: anti-mouse KLRG1 PE-Cy7 (2F1) , Thermo Fisher , Cat #25-5893-82; RRID: AB_1518768.

Techniques: Infection, Expressing, Staining

Journal: Cell reports

Article Title: PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8 + T cells at the expense of central memory

doi: 10.1016/j.celrep.2021.109804

Figure Lengend Snippet:

Article Snippet: anti-mouse KLRG1 PE-Cy7 (2F1) , Thermo Fisher , Cat #25-5893-82; RRID: AB_1518768.

Techniques: Purification, Control, Virus, Recombinant, Binding Assay, Protease Inhibitor, SYBR Green Assay, Staining, Cell Isolation, Software

Journal: NPJ Vaccines

Article Title: Evaluation of adenoviral vector Ad19a encoding RSV-F as novel vaccine against respiratory syncytial virus

doi: 10.1038/s41541-024-01001-z

Figure Lengend Snippet: Balb/c mice were immunized as described before and lymphocytes from lungs were examined 36 days post boost. Antigen experienced CD8 + T-cells were identified by F 85-93 -specific pentamer staining and intravascular (iv) CD45 pan staining. CD45-labelled cells (iv + ) were defined as circulating and iv protected (iv - ) as tissue resident memory cells. a The absolute number of F 85-93 + CD8 + with the relative contribution of iv + and iv - cells was summarized for each group. Statistical analysis was performed over whole population. b Within the iv + F 85-93 + CD8 + population central memory T-cells (T CM ; CD127 + KLRG1 - CD69 - CD103 - ), effector T-cells (T EFF ; CD127 - KLRG1 + ) and effector memory T-cells (T EM ; CD127 + KLRG1 + ) were determined, whereas within the iv - population tissue resident memory T-cells (T RM ; KLRG1 - CD103 + CD69 + or KLRG1 - CD103 + CD69 - or KLRG1 - CD103 - CD69 + ) were defined . Bars represent mean values with +SEM; naive n = 3, rAd5 (i.n.) n = 5, other groups n = 6. Data were analysed by one-way ANOVA followed by Tukey´s multiple comparison test. Statistically significant differences were indicated only among the used vaccine vectors within the intranasal resp. intramuscular group (* p < 0.05; ** p < 0.005).

Article Snippet: For the phenotypic T-cell analyses during the RSV challenge, the intravascular staining was omitted and the following panel was used for staining: anti-CD45-BV510 (1:500; 30-F11, BioLegend), anti-CD103-BV605 (1:100; 2E7, BioLegend), anti-CD8a-BV702 (1:200; 53-6.7, BioLegend), anti-CD127-FITC, anti-CD69-PerCP-Cy5.5 (1:200; H1.2F3, BioLegend), anti-CD44-PE (1:5000; IM7, BD Bioscience), anti-KLRG1-PE-Cy7 (1:300, 2F1, eBioscience) and Fixable Viablilty Dye eFluor® 780.

Techniques: Staining, Comparison

Journal: NPJ Vaccines

Article Title: Evaluation of adenoviral vector Ad19a encoding RSV-F as novel vaccine against respiratory syncytial virus

doi: 10.1038/s41541-024-01001-z

Figure Lengend Snippet: Balb/c mice were immunized and challenged as described in Fig. and lymphocytes from lungs were examined on respective days post-infection. Antigen experienced CD8 + T-cells were identified by CD44 + and F 85-93 -specific pentamer staining. a The absolute number of CD44 + F 85-93 + CD8 + T-cells was summarized for each group. Within the CD44 + F 85-93 + CD8 + T-cell population tissue resident memory T-cells (T RM ; KLRG1 - CD103 + CD69 + ) ( b ), central memory T-cells (T CM ; CD127 + KLRG1 - CD69 - CD103 - ) ( c ), effector memory T-cells (T EM ; CD127 + KLRG1 + ) ( d ) and effector T-cells (T EFF ; CD127 - KLRG1 + ) ( e ) were determined. Time points represent mean values with +SEM; all groups per time point n = 5. Data were analysed by two-way ANOVA followed by Dunnett´s multiple comparison test. Statistically significant differences were indicated among naive and vaccinated groups; (o: statistically significant worse than naive; +: statistically significant better than naive).

Article Snippet: For the phenotypic T-cell analyses during the RSV challenge, the intravascular staining was omitted and the following panel was used for staining: anti-CD45-BV510 (1:500; 30-F11, BioLegend), anti-CD103-BV605 (1:100; 2E7, BioLegend), anti-CD8a-BV702 (1:200; 53-6.7, BioLegend), anti-CD127-FITC, anti-CD69-PerCP-Cy5.5 (1:200; H1.2F3, BioLegend), anti-CD44-PE (1:5000; IM7, BD Bioscience), anti-KLRG1-PE-Cy7 (1:300, 2F1, eBioscience) and Fixable Viablilty Dye eFluor® 780.

Techniques: Infection, Staining, Comparison