Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Representative anamnestic vaccine-induced immune responses in the vaccinees. All vaccinees made CD8+ T-cell responses to several epitopes, including some restricted by the MHC-I molecule Mamu-A*02. In the interest of space, we are showing only two of the vaccinees in this figure; data from the remaining vaccinees are shown elsewhere (see Fig. S3 in the supplemental material). Whole-PBMC responses are indicated by blue bars, with responses postchallenge indicated in dark blue and responses observed immediately prior to challenge indicated in light blue. Responses observed in PBMCs depleted of CD8+ cells (red) are likely mediated by CD4+ T cells. CD8+ cell depletion was typically 99% complete (data not shown). Dark red bars indicate responses observed postchallenge, whereas light red bars indicate responses present immediately prior to the challenge. Green bars represent responses to minimal optimal peptides that bind to Mamu-A*02. As indicated in Table S1 in the supplemental material, some of these epitopes are conserved between SIVmac239 and SIVsmE660, whereas others have several substitutions, some of which could affect T-cell recognition. Again, light green bars indicate responses observed prior to the challenge, whereas dark green bars indicate anamnestic responses. Most anamnestic response analyses were performed at 14 to 15 days postinfection. For a couple of the animals (r00061, r02103), these assays were delayed to 21 days postinfection due to the very low viral loads observed.

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Frequency of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Breadth of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy

doi: 10.1172/JCI167417

Figure Lengend Snippet: ( A ) Brain tissue pieces were collected from the indicated brain regions and dissociated by mechanical disruption and enzymatic digestion. A CNS single-cell suspension was generated after Percoll separation. CD3 + T cells were positively selected and used for a CNS T cell QVOA. BrMCs and MG were isolated from the CD3 – fractions by CD11b + selection and by TMEM119 + selection, respectively. For human brains, CD11b + selection was performed to isolate BrMCs. BrMCs or MG at P0 (collected immediately after isolation) were used for purity and phenotype analysis and for RT-qPCR to measure proviral DNA and cell-associated RNA. BrMCs or MG at P1 were cultured 1–2 weeks ex vivo to allow the cells to recover and attach. P1 cells were used for the LRA study and the QVOA. ( B ) MG isolated from ART-suppressed, SIV-infected rhesus macaques were defined by TMEM119 staining (P1 MG) (scale bar: 100 μm) and ( C ) anti-TMEM119/anti-CD11b flow cytometry (P0 MG). ( D ) NHP MG proliferated ex vivo. ( E ) Total and integrated SIV DNA was detectable in isolated P0 MG ( n = 3). ( F ) SIV RNA was induced in isolated P1 MG 7 days after stimulation by the HDACi SAHA (500 nM), but was poorly induced by the canonical NF-κB agonist PEP005 (12 nM), the noncanonical NF-κB agonist AZD5582 (100 nM), or TNF-α (50 ng/mL). *** P < 0.001 compared with mock treatment, by 1-way ANOVA ( n = 3). ( G ) SIV RNA was recovered from the supernatant of NHP P1 MG cocultured with CEM174 ( n = 3). Data are presented as the mean ± SEM.

Article Snippet: For NHP samples, CD11b MicroBeads, NHP (catalog 130-091-100, Miltenyi Biotec) were used ( ).

Techniques: Disruption, Suspension, Generated, Isolation, Selection, Quantitative RT-PCR, Cell Culture, Ex Vivo, Infection, Staining, Flow Cytometry

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a , Comparison of SAMT-247 non-treated/treated effector cell-mediated ADCC activity in the vaccine ( n = 18) and vaccine + SAMT-247 groups ( n = 20; P < 0.0001). b , Correlation of SAMT-247-induced ADCC activity with number of intravaginal challenges in the vaccine + SAMT-247 group ( n = 20; P = 0.024). c , d , Intracellular Granzyme B, perforin, IFN‐γ and TNF-α in macaque rectal mucosal ( n = 9) NKG2A + cells in the presence or absence of different stimuli. e , Macaque rectal mucosal NKp44 + IL-17 + cells in the presence or absence of different stimuli ( n = 9). f , Correlation of efferocytosis with number of intravaginal challenges in animals in the vaccine group ( n = 18; P = 0.01). g , h , Comparison of percentage of efferocytosis ( P < 0.0001) ( g ) and efferocytosis MFI ( P < 0.0001) ( h ) using week 14 CD14 + monocytes in all vaccinated animals ( n = 38). i , Correlation of SAMT-247-induced efferocytosis (SAMT-247-untreated efferocytosis subtracted from SAMT-247-treated efferocytosis) with number of intravaginal challenges in the vaccine + SAMT-247 group ( n = 20; P = 0.065). Data shown in a , c , d , e , g and h were analysed with the two-tailed Wilcoxon signed-rank test. Data shown in b , f and i were analysed with the two-tailed Spearman correlation test. Horizontal and vertical bars denote mean and standard deviation, respectively.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Comparison, Activity Assay, Two Tailed Test, Standard Deviation

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a, b) Intracellular Granzyme B, perforin, IFN‐γ, and TNF-α in healthy human (n = 6) blood NKG2A + cells in the presence or absence of different stimuli. c ) Comparison of Env-specific rectal NKp44 + IL-17 + cells between vaccine+SAMT-247 (n = 20) and vaccine group (n = 18) 1 week post last vaccination ( P = 0.43). d ) Correlation of rectal mucosal Env-specific NKp44 + IL-17 + cells with number of intra-vaginal challenges in the vaccine group (n = 18). e ) Gating of NKG2A + NK cells, NKp44 + ILCs, and NKG2A – NKp44 – ILCs in rectal mucosal samples in the presence of PMA or PMA + SAMT-247 at 12 hours post stimulation. Gating was done on singlets, live, CD45 + , CD3 − , CD20 − , CD11b − cells. f ) Gating of NKp44 + IL-17 + ILCs in the rectal mucosal sample in the presence of PMA or PMA + SAMT-247 at 12 hours post stimulation. g ) Correlation of efferocytosis percentage with number of intra-vaginal challenges in the vaccine+SAMT-247 group (n = 20). ( h-i ) Comparison of h ) percentage of efferocytosis ( P < 0.0001) and i ) efferocytosis MFI ( P < 0.0001) using pre CD14 + monocytes in all vaccinated animals (n = 38). Data shown in ( a, b, h, i ) were analyzed with the two-tailed Wilcoxon signed-rank test or two-tailed Mann-Whitney test. Data shown in ( d, g ) were analyzed with the two-tailed Spearman correlation test.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a , Representative imaging of human NKG2A + cells unstimulated or stimulated with SAMT-247, PMA or PMA + SAMT-247. b , Mean zinc intensity in NKG2A + cells of the healthy human donor in the presence or absence of zinc chelator in different stimulation conditions ( n = 8). Fluorescence intensity of each field was measured for zinc expression as indicated by green colour, and the total number of DAPI positive cells were counted to determine the mean intensity of zinc/cells using iMARIS software. The mean of two duplicate fields was evaluated for the calculation. c , Comparison of expressions of NKG2A marker in macaques in the absence or presence of zinc chelator and stimuli in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). d – g , Comparison of expressions of granzyme B, perforin, IFN‐γ and TNF-α by macaque blood NKG2A + cells from week 17 in the absence or presence of different stimulations and zinc chelator in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). h , i , Evaluation of the frequency of CD14 + monocytes and CD14 + IL-10 + monocytes in the absence or presence of zinc chelator and stimuli in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). Data shown in b – i were analysed with the two-tailed Wilcoxon signed-rank test. Horizontal and vertical bars denote mean and standard deviation, respectively.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Imaging, Fluorescence, Expressing, Software, Comparison, Marker, Two Tailed Test, Standard Deviation

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: Vaccination-induced ADCC results in apoptosis of SIV-infected cells, which in turn are cleared by efferocytes to avoid inflammation and preserve tissue homeostasis. Vaccine-induced IL-10 expression in CD14 + monocytes further augments efferocytosis. Vaccine-induced NKp44 + cells produce the IL-17 cytokine that maintains mucosal epithelium integrity. All of these protective effector responses were enhanced dramatically in the vaccine + SAMT-247 group, increasing protection from SIV mac251 acquisition. The scheme is adapted from Bissa et al. .

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Infection, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

doi: 10.1136/jitc-2024-010239

Figure Lengend Snippet: 15–4P Stimulation increases NKG2D expression and murine NK cell proliferation compared with IL-15/IL-15Rα stimulation alone. ( A ) Schematic showing AgN2a-4P and NK cell receptor-ligand interactions. B6 NK cells are isolated and cultured with IL-15/IL-15Rα conjugate and with or without irradiated AgN2a-4P cells at a 1:1 ratio for 12 days. All expression markers were examined on CD3 − NK1.1 + NK cells. ( B ) Dot plots of NKG2D (top) and NKp46 (bottom) on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with percentage of gated cells. ( C ) Per cent of NK cells positive for NKG2D (left) and NKp46 (right) and MFI below. ( D ) Fold change of IL-15 NK cells and 15–4P stimulated NK cell expansion at day 12 compared with day 0. ( E ) Dot plots of proliferation marker Ki-67 on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with ( F ) percentage and MFI. ( G ) Percentage and MFI of TRAIL (top) and Fas-L (bottom) were assessed for each group. Data are representative of an experiment that was replicated two times. Representative dot plots examples of one replicate out of a minimum of three replicates. All bar graphs show individual experimental replicates plotted with mean and SEM (n=3–5). Two-sided two-sample t-tests were performed where indicated (*p<0.05, **p<0.01, ***p<0.001, ns=not significant). AgN2a, aggressive variant of the Neuro-2a murine neuroblastoma cell line; MFI, median fluorescence intensity; NK, natural killer.

Article Snippet: T cells were depleted using a CD3 MicroBead Kit (Miltenyi Biotec) by negative selection on the AutoMACs Pro Separator (Miltenyi Biotec) at day+0.

Techniques: Expressing, Isolation, Cell Culture, Irradiation, Marker, Variant Assay, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Chronic SIV-Induced neuroinflammation disrupts CCR7 + CD4 + T cell immunosurveillance in the rhesus macaque brain

doi: 10.1172/JCI175332

Figure Lengend Snippet: ( A – C ) Schematic of single CD45 + cell profiling in brain, right hemisphere (RH) and spleen. ( D ) Differences in B and T cell transcripts in brain versus spleen. ( E ) UMAP of scRNA-Seq transcriptional profiles from brain and spleen identifies 10 clusters. Cell clusters are color-coded based on cell identity assigned using Single R. SkM, skeletal muscle; MBC, memory B cells; Mono, monocytes. Inset shows cell proportions in each cluster split by tissue type (bottom, spleen; top, brain). ( F ) UMAP shows 10 subclusters from T cell clusters in E .

Article Snippet: Cells were washed in complete medium and CD45 + cells were isolated using CD45 microbeads for nonhuman primates (Miltenyi Biotech) in accordance with the manufacturer’s protocol.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Chronic SIV-Induced neuroinflammation disrupts CCR7 + CD4 + T cell immunosurveillance in the rhesus macaque brain

doi: 10.1172/JCI175332

Figure Lengend Snippet: ( A ) Representative illustration for ROI selection within the hippocampal region of control (top) and SIVCL757-infected (bottom) animals; Nuclear (blue), CD3 (green), CD45 (red), and NeuN (purple) for Nanostring whole transcriptome analysis (WTA) and proteomics pipeline. ( B ) CD3ε mRNA and protein counts for ROIs. ( C ) Protein counts for all ROIs. ( D ) Differentially expressed neurodegenerative genes across control and SIV-infected ROIs. ( E ) Differentially expressed metabolic genes across control and SIV-infected ROIs. ( F ) UMAP plot shows cell annotation for myeloid specific gene clusters from sc data. Dot plots depict average gene expression of canonical microglia, monocyte, macrophage, antiviral, and inflammatory response genes across 8 distinct myeloid clusters. ( G ) Chord plot of differentially expressed genes across control and SIV-infected CD45-enriched cells from sc transcriptomics. Genes related to TCR signaling pathway are colored in green for clarity. *** P < 0.001.

Article Snippet: Cells were washed in complete medium and CD45 + cells were isolated using CD45 microbeads for nonhuman primates (Miltenyi Biotech) in accordance with the manufacturer’s protocol.

Techniques: Selection, Control, Infection, Gene Expression