peptide pool from cmv  (Mabtech Inc)

Journal: bioRxiv

Article Title: An early functional adaptive NK cell signature drives optimal CD8 + T-cell activation and predicts sustained HIV-1 viral control

doi: 10.1101/2025.03.17.643703

Figure Lengend Snippet: (A) Representative flow plots and (B) summary data for the frequency of IFN-γ-producing CD56 dim NK cells following 6-hour of stimulation with antibody-coated Raji cells (Target cells) in individuals with subtype A and non-A subtype infection. Control = Uncoated cells. (C) Representative flow plots and (D) pie charts showing IFN-γ production in FcεRγ + CD57 + CD56 dim and FcεRγ − CD57 + CD56 dim NK cells in participants with subtype A and non-A subtype infection. (E) The frequencies of IFN-γ + FcεRγ − CD57 + CD56 dim NK cells (dashed line) and log 10 viral loads (grey solid line) at all time points. (F) Representative plots for the identification of antigen-reactive NK cells based on double expression (IFN-γ and CD107a) following 6-h stimulation with media alone (control) or with HIV-1 (Gag and Env) or CMV (pp65) peptide pools. (G) a Percentage of responders (subtype A) to HIV-1 peptide pools at each time point or CMV pools at 1-month time point. (H) Frequency of HIV-reactive NK cells at all time points. Pie charts representing the proportion of antigen-reactive NK cells within adaptive (NKG2C + ) or canonical (NKG2C − ) NK cells. Significance determined by two-tailed Mann– Whitney U test or Wilcoxon matched-paired signed rank test. *p < 0.05, **p < 0.01. The non-parametric Spearman test was used for correlation analysis (two-tailed).

Article Snippet: After overnight rest, PBMCs were stimulated for 6 hours with 2 μg/ml of cohort-specific HIV-1 Gag or ENV peptide pools (provided by IAVI) or cytomegalovirus (CMV)-pp65 peptide pools (Miltenyibiotec) in the presence of αCD28/αCD49d co-Stim antibodies (1 μg/ml), GolgiStop (containing Monensin, 2 μmol/l), GolgiPlug (containing brefeldin A, 10 μg/ml) (BD Biosciences) and anti-CD107α BV421 antibody (BD Biosciences, Catalog # 562623, dilution 1 in 200).

Techniques: Infection, Control, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Distinctive CD8 + T cell activation by antigen-presenting plasmacytoid dendritic cells compared to conventional dendritic cells.

doi: 10.1016/j.celrep.2025.115413

Figure Lengend Snippet: Figure 4. cHSPC-pDCs can cross-present antigen to CD8+ T cells (A) moDCs (blue) and cHSPC-pDCs (lilac) were cultured +/ long CMV peptide (CMVpep-long) and +/TLR4 agonist for moDCs or +/ TLR7 agonist for pDCs. Autologous CD8+ T cells were added (ratio T/DC 10:1) and IFN-g secretion measured after 24 h (n = 3, in duplicate). (B) IFN-g secretion by CD8+ T cells after co-culture with R837 + CMVpep-long-stimulated cHSPC-pDCs with isotype control (isot ctrl) or anti-HLA-ABC blocking antibody (aHLA-ABC, n = 3, in duplicate). (C) Western blot of human fibroblast +/ CMV infection (CMV pp65) and vinculin (VCL) as loading control. (D) Cells cultured as described in (A) but, instead of peptide, cultured with cell lysate from CMV-infected fibroblasts (CMVlysate), and IFN-g secretion was quantified (n = 3, in duplicate). (E) IFN-g secretion by CD8+ T cells after co-culture with R837 + CMVlysate-stimulated cHSPC-pDCs with isotype control (isot ctrl) or anti-HLA-ABC blocking antibody (aHLA-ABC, n = 3, in duplicate). (F) Experimental outline. First PBMC isolation: CD34+ collection and differentiation into cHSPC-pDCs. Second PBMC isolation, 2 weeks after the first draw: mDC enrichment, and some PBMCs kept in culture with IL-2 for CD8+ T cell enrichment. Both cHSPC-pDCs and mDCs were cultured +/ CMV peptides and +/ TLR7 and +/ TLR4 agonist. DCs were co-cultured with autologous CD8+ T cells (DC/T cell 1:10), and T cell activation was determined by IFN-g secretion. (G) IFN-g secretion by CD8+ T cells after co-culture with mDCs (green) or cHSPC-pDCs (lilac, n = 3, in duplicate). Error bars represent mean ± SEM and symbols individual donors. Equal symbols represent equal donors. Statistical significance was determined using the one-way ANOVA multiple-comparisons test (A, D, and E) and Wilcoxon matched-pairs signed-rank test (B and E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains TB40EIEr reporter variant of HCMV strain TB40E (GenBank accession number EF999921) Chemicals, peptides, and recombinant proteins human Flt3-L Peprotech 300–19 human TPO Peprotech 300-18) human SCF Peprotech 300–07 human IL3 Peprotech 200-03) SR1 StemCell Technologies 72344) human IFNb PBL Assay Science 11410–2 human IFN-g Peprotech 300-02) human IL4 Peprotech 200–04 human GM-CSF Peprotech 300–03 human IL2 Peprotech 200–02 ProMixTM CMV Peptide Pool ProImmune PX-CMV phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich 79346 ionomycin Sigma-Aldrich I9657 R837 (Imiquimod) Invivogen Tlrl-imqs-1 CpG-A ODN 2216 Invivogen Tlrl-2216 LPS Sigma-Aldrich L2637 poly(I:C) Invivogen tlrl-picm Custom made CMV long peptide pool: Schafer-N ApS NA CMV_pp65_HRGPQYSEHPTFTSQYYRIQG-OH Schafer-N ApS NA CMV_pp65_HAGILARNLVPMVATVQGQNL-OH Schafer-N ApS NA CMV_pp65_HTVELRQYDPVAALFFFDIDL-OH Schafer-N ApS NA CMV_pp65_H-EPMSIYVYA LPLKMLNIPSI-OH Schafer-N ApS NA CMV_pp65_HQPFMRPHERNGFTVLCPKNM-OH Schafer-N ApS NA CMV_pp65_HTTERKTPRVTGGGAMAGAST-OH Schafer-N ApS NA CMV_pp65_HNLKYQEFFWDANDIYRIFAE-OH Schafer-N ApS NA CMV_pp65_HKMLNIPSINVHHYPSAAERK-OH Schafer-N ApS NA

Techniques: Cell Culture, Co-Culture Assay, Control, Blocking Assay, Western Blot, Infection, Isolation, Activation Assay