Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Comparison of Th17 cells, Treg cells, and Th17/Treg ratios between stroke patients and HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques: Comparison

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 was positively associated with a Th17/Treg imbalance. Association of serum RBP4 with Th17 cells (A) , Treg cells (B) , and the Th17/Treg ratio (C) in stroke patients. Association of serum RBP4 with Th17 cells (D) , Treg cells (E) , and the Th17/Treg ratio (F) in HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were negatively associated with the MMSE score at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with MMSE score at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were associated with the occurrence of cognitive impairment at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with cognitive impairment at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Serum CD95L in SLE Patients Induces Transmigration of T Lymphocytes (A) Soluble CD95L levels were measured in serum from newly diagnosed SLE patients (n = 34) and healthy donors (n = 8) via ELISA (Student’s t test). (B) CD95L in SLE serum was fractionated by size-exclusion column chromatography and measured via ELISA. Inset : CD95L was immunoprecipitated from gel-filtration fractions 40–46 and 76–78 and subjected to immunoblotting. The image is representative of gel-filtration analysis of four different patients. (C) CD95L and IL-17 staining in inflamed skin samples from lupus patients or healthy mastectomy subjects. Numbers represent different patients. The scale bar represents 100 μm. (D) CD95L, CD31, CD4, and IL-17 staining in inflamed skin samples from an SLE patient. “V” represents an endothelial vessel, and arrowheads identify marker-expressing cells. (E) Densitometric analysis of CD95L and IL-17 staining in different patients. (F) Transmigration of human T cell subpopulations in the presence of serum from SLE patients or healthy donors. (G) Th17 T cell transmigration in the presence of SLE serum containing the indicated concentrations of CD95-Fc. Undifferentiated Th 0 T cells served as controls. Data represent means ± SD of five individual serum donors. (H) CD4 + T cell transmigration with or without cl-CD95L (100 ng/mL). Data represent means ± SD of three independent experiments. (I) Treg and Th17 cell transmigration with or without cl-CD95L (100 ng/mL) Data represent means ± SD of three independent experiments (two-way ANOVA).

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Transmigration Assay, Enzyme-linked Immunosorbent Assay, Column Chromatography, Immunoprecipitation, Filtration, Western Blot, Staining, Marker, Expressing

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Transcriptomic Signature in Human Th17 and Treg Cells Stimulated with cl-CD95L (A) Venn diagram comparing the genes differentially expressed between untreated and cl-CD95L-treated T cell subsets. (B) Heat map depicting the relative n-fold change in the amount of transcripts significantly (p ≤ 0.05) and differentially expressed between Th17 and Treg cells stimulated with cl-CD95L (100 ng/mL). Data for each experimental group (n = 2 per condition) are shown. The color gradient indicates n-fold change, as shown. (C) Pathway enrichment analysis of genes whose expression is significantly modulated by cl-CD95L in Th17 and Treg cells and associated p values. (D) Left panel : endothelial transmigration of human Th17 and Treg cells was evaluated in the presence or absence of cl-CD95L (100 ng/mL) in the Boyden chamber. Right panel : Th17 cells were pre-treated with FTY720 (1 μM), TY-52156 (10 μM), VPC-23019 (10 μM), W146 (1 μM), or CAY-1044 (1 μM) and then exposed to cl-CD95L (100 ng/mL), and endothelial transmigration was evaluated via the Boyden chamber. Data represent means ± SD of three independent experiments.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Expressing, Transmigration Assay

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Cl-CD95L Is a Chemoattractant for Th17 Cells In Vivo Mice received a single injection of cl-CD95L (200 ng/animal) or vehicle and were sequentially sampled. (A) T cell populations were obtained from tissues or peritoneal cavity washes. Cells were then restimulated in the presence of PMA and ionomycin for 4 hr and then analyzed by flow cytometry. Cells were identified as follows; Th1 (CD4 + IFN-γ + ), Th17 (CD4 + IL-17 + ), and Treg (CD4 + Foxp3 + ). Numbers of infiltrating cells were calculated. (B) Ratios of Th17/Th1 cells per organ were determined 24 hr after injection. Data represent two independent experiments with six mice/group; means ± SEM are displayed.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: In Vivo, Injection, Flow Cytometry

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: CD95 Induces a DD-Independent Ca 2+ Response (A) CEM T cells were stimulated with cl-CD95L (100 ng/mL). Cells were lysed, and CD95 was immunoprecipitated. The protein complex was resolved by SDS-PAGE and subjected to immunoblotting. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments. (B) Left panel : Th17 cells from peripheral blood were stimulated with cl-CD95L (100 ng/mL) for indicated times. PLA was performed with anti-CD95 and anti-PLCγ1 mAbs. Nuclei were stained in blue (DAPI). Red dots were observed when the distance between anti-CD95 and anti-PLCγ1 mAbs was close (≈16 nm). Right panels : Red dots were counted in 200 cells taken from different fields. Data represent means ± SD of three independent experiments. (C) Schematic diagram of CD95 constructs. (D) CEM-IRC cells expressing GFP alone or the GFP-fused CD95 constructs shown in (C) were loaded with the Ca 2+ probe, Fluo2-AM (1 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow), and the intracellular calcium concentration ([Ca 2+ ] i ) was monitored. Data are given as means ± SD of three experiments performed independently on n = 20 cells. (E) HEK cells transfected with the indicated constructs were stimulated with CD95L (100 ng/mL) for indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments. (F) Activated PBLs were pre-incubated for 1 hr with TAT-control or TAT-CID (10 μM) and stimulated with cl-CD95L (100 ng/mL) for the indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates a treatment with beads alone. Data are representative of three independent experiments. (G) Activated PBLs from healthy donors were loaded with FuraPE3-AM (1 μM) and pre-treated for 1 hr with TAT-control or TAT-CID (10 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow). Data represent means ± SD.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Staining, Construct, Expressing, Concentration Assay, Transfection, Incubation, Control

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: TAT-CID Alters Immunological Parameters in Lupus-Prone Mice (A) Transmigration of mouse Th17 cells was monitored with the indicated concentrations of TAT-CID. (B–D) C57BL/6 mice were injected with TAT-control or TAT-CID (40 mg/kg) 2 hr prior to intraperitoneal injection of cl-CD95L (200 ng) or vehicle. Animals were examined 24 hr after cl-CD95L injection. (B) Total cell counts in the peritoneal cavity are shown. (C) PECs were subjected to magnetic bead separation to identify the percentage of infiltrating CD4 + CD62L − (activated) T cells. (D) IL-17A concentrations in the peritoneal cavity were measured via ELISA (two-way ANOVA). Data in (B)–(D) represent two independent experiments performed with six mice/group. Data are means ± SEM. (E–I) MRL. Fas lpr/+ mice received either TAT-CID or TAT-control for 5 weeks. (E) Upon completion of the experimental protocol, ratios of spleen weight to body weight of individual animals were measured and compared to those of age-matched MRL and homozygous MRL. Fas lpr/lpr mice. (F) Total cell number in the spleen is shown. (G) Cellular composition of the spleen was determined in regard to the number of CD4 + CD62L − T cells. (H) mRNA expression levels of il-23r, ccr6 , and ror-γt in cells from (C). (I) Isolated T cells from (G) were re-stimulated with anti-CD3 mAb for 72 hr. IL-17A was then quantified by ELISA (unpaired Student’s t test).

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Transmigration Assay, Injection, Control, Enzyme-linked Immunosorbent Assay, Expressing, Isolation

Journal: Human Reproduction Update

Article Title: The endometrial immune environment of women with endometriosis

doi: 10.1093/humupd/dmz018

Figure Lengend Snippet: Schematic of interactions of immune populations in normal (A) and endometriosis eutopic endometrium with possible effects in ectopic sites (B). In normal conditions, Mø are involved in clearing cell debris during menses, tissue regeneration and angiogenesis. In addition, Mø1 have pro-inflammatory properties and Mø2 have anti-inflammatory properties. It has been described that Mø2 predominate in normal endometrium, indicating that eutopic endometrium in healthy women has an anti-inflammatory environment, which allows embryo implantation. In endometriosis, it has been suggested that the main population of Mø is Mø1, indicating that the environment is more pro-inflammatory, which affects embryo implantation. In addition, Mø do not increase in the secretory phase, as well as DC, which implies that cell debris can survive and migrate to the peritoneal cavity, implant and develop the ectopic lesions. In addition, Treg cells also promote an anti-inflammatory environment in normal eutopic endometrium. However, they are deregulated in women with endometriosis, which leads to a more pro-inflammatory environment. Another important aspect to consider is that NK are less cytotoxic in women with endometriosis, which affects embryonic implantation. In general, we can observe that in endometriosis eutopic endometrium, the anti-inflammatory functions are inhibited by the aberrant function of some immune populations. In addition, this affects the survival of endometrial cells, which can migrate to ectopic sites and develop the endomeriotic lesions. The peritoneal cavity could initially be an anti-inflammatory environment (increase of Mø2, Treg cells and Th2 and defective NK), allowing the implantation of endometrial cells in the ectopic sites. Upon lesion development, activation of pro-inflammatory immune populations, such as MC, NT, EN, Th1 and Th17, may occur, allowing the maintenance of the endometriotic lesion by promoting angiogenesis, fibrotic adhesions and an hospitable inflammatory environment preventing other immune cells from clearing ectopic lesions. However, all these interactions between immune cells need further investigation. Abbreviations: Mø1, macrophages 1; Mø2, macrophages 2; DC, dendritic cells; NK, natural killer cells; MC, mast cells; EN, eosinophils; NT, neutrophils; B cells, B lymphocytes; T cells, T lymphocytes; T helper cells (Th1, Th2 and Th17); Treg cells, regulatory T cells.

Article Snippet: CD4+ Th17 , Fluctuation menstrual cycle: Increased in proliferative phase ( Mettler et al., 1996 ) Function: Fighting against infections , Fluctuation menstrual cycle: Not described Function: Not described Comparison with normal eutopic endometrium: Not described , Fluctuation menstrual cycle: Not described Function: Involved in growth lesion Comparison with normal: Increased CD4+ Th17 cells in peritoneal fluid and blood compared to controls ( Gogacz et al ., 2016 )/No variation in serum or blood ( Andreoli et al., 2011 ).

Techniques: Activation Assay

Journal: Human Reproduction Update

Article Title: The endometrial immune environment of women with endometriosis

doi: 10.1093/humupd/dmz018

Figure Lengend Snippet: Markers of immune cell populations described in endometrium .

Article Snippet: CD4+ Th17 , Fluctuation menstrual cycle: Increased in proliferative phase ( Mettler et al., 1996 ) Function: Fighting against infections , Fluctuation menstrual cycle: Not described Function: Not described Comparison with normal eutopic endometrium: Not described , Fluctuation menstrual cycle: Not described Function: Involved in growth lesion Comparison with normal: Increased CD4+ Th17 cells in peritoneal fluid and blood compared to controls ( Gogacz et al ., 2016 )/No variation in serum or blood ( Andreoli et al., 2011 ).

Techniques: Activation Assay

Journal: Human Reproduction Update

Article Title: The endometrial immune environment of women with endometriosis

doi: 10.1093/humupd/dmz018

Figure Lengend Snippet: Immune cell populations in endometrium and endometriosis .

Article Snippet: CD4+ Th17 , Fluctuation menstrual cycle: Increased in proliferative phase ( Mettler et al., 1996 ) Function: Fighting against infections , Fluctuation menstrual cycle: Not described Function: Not described Comparison with normal eutopic endometrium: Not described , Fluctuation menstrual cycle: Not described Function: Involved in growth lesion Comparison with normal: Increased CD4+ Th17 cells in peritoneal fluid and blood compared to controls ( Gogacz et al ., 2016 )/No variation in serum or blood ( Andreoli et al., 2011 ).

Techniques: Migration, Comparison, Activity Assay, Activation Assay, Concentration Assay, Chemotaxis Assay

Journal: PLoS Pathogens

Article Title: Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

doi: 10.1371/journal.ppat.1004543

Figure Lengend Snippet: To quantify the expression of Il-17 and IL-22 in CD4+T cells, mucosal and peripheral mononuclear cells were stimulated for 5 hours with 40 ng/mL PMA and 1 µM Ionomycin. Gating strategy of sigmoid colon Th17 and Th22 CD4+T cells is shown for a FI subject ( a ) and representative flow cytometry plots including unstimulated controls (unstim) gated on CD4+T cells showing expression of IL-17 and/or IL-22 in FI ( b ), FIII ( c ) and FV ( d ) in the sigmoid colon. A decrease in the frequency of IL-17 ( e ), IL-22 ( f ), IL-17/IL-22 ( g )-producing cells was seen as well as in the subpopulation of triple-cytokine producing (IL-2, IL-22, IFNγ) Th17 cells ( h ) from FI (black circle)/FII (red circle) to FIII and FIV (red square)/FV (black square). Frequency of single and double cytokine producing cells is calculated from percentage CD4+T cells, while triple-cytokine producing cells are calculated from percentage of CD4+IL17+T cells. All comparisons were made to FI/II: *p≤0.05, **p≤0.01 and ***p≤0.001.

Article Snippet: There was a significantly lower proportion of Th17 cells in peripheral blood (HIV-: 0.37%, FI/II: 0.65%, FIII: 0.5% and FIV/V: 0.86%, CHI: 0.48%) compared to the sigmoid colon with no quantitative changes observed between Fiebig stages.

Techniques: Expressing, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

doi: 10.1371/journal.ppat.1004543

Figure Lengend Snippet: Subjects that initiated ART during FI (black circle)/FII (red circle) for 6 months were able to maintain mucosal CD4+T cells ( a ), CD4+CCR5+ ( b ), Th17 cells ( c ), triple cytokine-producing Th17 cells ( d ), Th22 cells ( e ), and IL-17 and/or IL-22 ( f ) producing CD4+T cells with no differences when compared to HIV-. In addition their CD8 activation in the mucosa ( g ) and periphery ( h ) normalized after 6 months of ART. Frequency of single and double cytokine producing cells was calculated from percentage CD4+T cells, while triple-cytokine producing cells were calculated from percentage of CD4+IL17+T cells. *p≤0.05, **p≤0.01 and ***p≤0.001; DR: HLA-DR; blue dotted line: median of HIV- individuals; purple dotted line: median of CHI individuals.

Article Snippet: There was a significantly lower proportion of Th17 cells in peripheral blood (HIV-: 0.37%, FI/II: 0.65%, FIII: 0.5% and FIV/V: 0.86%, CHI: 0.48%) compared to the sigmoid colon with no quantitative changes observed between Fiebig stages.

Techniques: Activation Assay

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: Two new subsets of memory CD4 + T-cells express Th17 lineage markers. a Memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) isolated from the peripheral blood of HIV-uninfected individuals were analyzed for their differential expression of CCR6, CCR4, and CXCR3. CCR6 + subsets included: CCR4 + CXCR3 − (Th17), CCR4 − CXCR3 − (double positive, CCR6 + DP), CCR4 + CXCR3 + (double negative, CCR6 + DN), and CCR4 − CXCR3 + (Th1Th17). CCR6 − subsets included: CCR4 + CXCR3 − (Th2), CCR4 + CXCR3 + (CCR6 − DP), CCR4 − CXCR3 − (CCR6 − DN) and CCR4 − CXCR3 + (Th1). Shown is the frequency of CCR6 + and CCR6 − subsets ( b ; n = 30) and their expression of CD161 ( c ; n = 8). Each symbol represents a distinct subject. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. d Shown are median frequencies of central (CM, CCR7 + CD27 + ), transitional (TM, CCR7 − CD27 + ) and effector (EM, CCR7 − CD27 − ) memory cells per CCR6 + subset (n = 10). e – g FACS-sorted memory subsets (S1 Figure) were stimulated via CD3/CD28 for 4 days. e The production of the lineage-specific cytokines IL-17A, IFN-γ, and IL-5 was quantified by ELISA. Shown are results (mean ± SEM) on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6- (n = 3–7). Paired t Test p -values are indicated on the figures . f Transcriptional profiling were generated using the HumanHT-12 v4 Expression BeadChip; (Illumina). The heat map depicts differential expression of well-established Th17 and Th1 transcripts (identified as being up/down regulated in Th17 versus CCR6 − DN, p value <0.05, fold change cut-off 1.3) in matched CCR6 − DN versusCCR6 + DN, CCR6 + DP and Th17 (n = 4–6; up-regulated genes in red; down-regulated genes in blue )

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Isolation, Quantitative Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Generated

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: CCR6 + DN and CCR6 + DP cells express unique transcriptional signatures. Genome-wide transcriptional profiling was performed on sorted matched Th17, CCR6 + DN, and CCR6 + DP (n = 4–6) isolated from the peripheral blood of HIV-uninfected individuals, as in Fig. f. Shown are ( a ) volcano representation of differentially expressed probe sets in CCR6 + DN versus Th17, CCR6 + DP versus Th17, and CCR6 + DN versus CCR6 + DP (depicted in red : p values <0.05 and fold change cut-off 1.3). b , d For the same contrasts, shown are heat maps depicting top modulated pathways identified using Ingenuity Pathway Analysis (IPA) ( b ) and Gene Set Variation Analysis (GSVA) canonical pathways ( c ) and biological functions ( d ). Genes up and down regulated in different subsets are represented in red and blue, respectively. e Expression of STAT3, BCL6, LEF1, and TERC mRNA was quantified by real-time RT-PCR (mean ± SEM; n = 3–4). Paired t-Test p -values are indicated on the figures

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Genome Wide, Isolation, Expressing, Quantitative RT-PCR

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: CCR6 + DN cells are a major source of IL-17F, IL-8, and IL-21. Culture supernatants harvested from Th17, Th1Th17, and CCR6 + DN (stimulated as in Fig. e) isolated from the peripheral blood of HIV-uninfected individuals were screened for the expression of 34 T-helper lineage-specific cytokines using the Human Th1/Th2/Th17 Antibody Array C series (RayBiotec). a , b Shown are results from one experiment with matched Th17 versus CCR6 + DN and Th1Th17 versus CCR6 + DN subsets: membrane blot ( left panels ) and relative density quantification ( right panels ). Results are representative of experiments performed with cells from two different donors: c , d Levels of IL-17F, IL-22, CCL20, IL-10, IL-13, TNF-α, IL-8, and IL-21 were quantified by ELISA. Shown are results on matched Th17, Th1Th17, CCR6 + DN, CCR6 + DP, and CCR6 − samples from different individuals (n = 3–7, mean ± SEM). Paired t -Test p -values are indicated in the graphs

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Isolation, Expressing, Ab Array, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: CCR6 + DN distinguish from the other Th17-subsets by superior frequency/counts in CI on ART individuals. a PBMCs from CI on ART individuals (n = 20; Additional file : Table S2) were stained as in Fig. . Shown is the relative frequency of the four CCR6 + subsets in CI on ART individuals. Paired t-Test p -values are indicated on the figures . Horizontal bars indicate median values. b Shown are median frequencies of CM, TM, and EM within each CCR6 + subsets from CI on ART (n = 10). Shown is the frequency ( c ) and counts ( d ) of the four CCR6 + subsets in CI on ART versus uninfected individuals. Cell counts were calculated taking into account their frequency within the total CD4 + T-cell fraction. e The dynamics of CCR6 + subset counts were investigated longitudinally in n = 5 HIV-infected individuals from the Montreal HIV Primary infection cohort (Additional file : Table S3), in relationship with plasma viral load, before and after ART initiation ( grey ). f Shown are statistical analysis for differences in cell counts between the four CCR6 + subsets (n = 5 HIV-infected individuals) using Friedman test and the post-test Dunn’s multiple comparison and Wilcoxon t test

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Staining, Infection, Clinical Proteomics, Comparison

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: CCR6 + DN are predominant in lymph nodes of HIV-infected individuals receiving ART. a , b Matched PBMCs and inguinal lymph node cells from three CI on ART individuals (CI 36, CI 37, CI 38; Additional file : S2 Table) were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR7 Abs. A viability staining was used to exclude dead cells. Viable memory CD4 + T-cells (CD3 + CD4 + CD45RA − ) expressing CCR6 were analyzed for their differential expression of CCR4 and CXCR3. The four CCR6 + subsets including Th17, CCR6 + DP, CCR6 + DN, and Th1Th17 were identified in both PBMCs and cells from lymph nodes. a Shown is the phenotype of PBMCs ( upper panels ) and lymph node cells ( lower panels ) in one representative donor. b Shown are statistical analysis of the frequency of CCR6 + subsets in the lymph node (n = 3). Paired t-Test p -values are indicated on the figures

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Infection, Staining, Expressing, Quantitative Proteomics

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: CCR6 + DN and CCR6 + DP subsets are permissive to HIV infection in vitro. a PBMCs from healthy individuals were stained with a cocktail of fluorochrome-conjugated CD3, CD4, CD45RA, CCR4, CXCR3, CCR6, and CCR5 or CXCR4 Abs. The gating strategy for the identification of distinct CCR6 + and CCR6 − T-cell subsets was designed as in Fig. a. The frequency of cells expressing CCR5 ( left panel ) and CXCR4 ( right panel ) was analyzed within the Th17, Th1Th17, CCR6 + DN and CCR6 + DP subsets. Paired t-Test p -values are indicated on the figures. Horizontal bars indicate median values. b Memory CCR6 + subsets from three HIV-uninfected subjects were sorted and stimulated via CD3/CD28 for 4 days, as in Fig. e. Cells were exposed to a highly infectious R5 strain HIV-ADA8. Levels of HIV-p24 were quantified by ELISA in cell supernatants at day 3 posy-infection

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Infection, In Vitro, Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Retrovirology

Article Title: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

doi: 10.1186/s12977-016-0293-6

Figure Lengend Snippet: New insights into the heterogeneity of human Th17 cells at homeostasis and during ART-controlled HIV-1 infection. In this work we identified two new subsets of CCR6 + T-cells, CCR6 + DN/CCR4 − CXCR3 − and CCR6 + DP/CCR4 + CXCR3 + , that share Th17 features with the previously described Th17/CCR4 + CXCR3 − and Th1Th17/CCR4 − CXCR3 + . Despite these similarities, CCR6 + DN distinguished from the other three subsets by superior their ability to produce Th17 effector cytokines ( e.g., IL-17F, IL-8, and IL-21) and their predominant frequency/counts in the blood and lymph nodes HIV-infected individuals receiving ART. Finally, we demonstrate that CCR6 + DN harbor replication-competent HIV-DNA. Thus, we reveal the existence in humans of four Th17-polarized CCR6 + subsets that represent distinct stages of Th17 differentiation, with CCR6 + DN being the most predominant and contributing to HIV reservoir persistence under ART

Article Snippet: Recent evidence from our group (Gosselin et al, unpublished observations) and others [ ], support the contribution of long-lived Th17 cells to HIV reservoir persistence under ART.

Techniques: Infection