Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Expression and T cell Regulatory Action of the PD-1 Immune Checkpoint in the Ovary and Fallopian Tube

doi: 10.1111/aji.13649

Figure Lengend Snippet: Brown stain indicates expression. PD-1 was detected in some oocytes (compare faintly positive examples in panel a with negative example in b; region from white box in panel d is shown in panel a), and granulosa cells of large antral follicles (c, arrow; theca interna denoted by *). PD-1 was present at high levels in relatively rare cells throughout the ovary (red arrowheads in insets, b and d) and the stromal compartment of the fallopian tube (inset e’, *); some of these are likely to be T cells and macrophages (see Figure S2). PD-1 was also detectable at low levels in the luminal epithelium of the fallopian tube (e and e’, arrow). PD-L1 was consistently expressed in oocytes (f, g) of all sizes, pregranulosa cells of non-growing follicles (f, arrow) and granulosa cells (f-h, arrows) and the theca interna (g, h *) of growing follicles. PD-L1 was also broadly expressed in stromal cells of the inner ovarian cortex (i, l - left), but was not detectable in cells of the OSE or in stromal layers immediately below the surface (i, red bracket and *’s; compare to Figs. 2e–g). PD-L1 was also expressed the luminal epithelial cells of the fallopian tube (j). PD-L2 was detected only in the granulosa cells (arrow) and theca interna (*) of large antral follicles (m, compare to panels c and h). Control circulating immune cell expression of PD-1 and ligands is shown in Supplemental Fig. S1’a–c, representative no first antibody control shown in Fig. S1’d.. A - follicular antrum, Ex - extra-ovarian space, IL - intraluminal space of tube, r - red blood cells. Scale bars in panels a, e, g, k, m and q = 100 μm, bars in other panels are 50 μm

Article Snippet: Where specified, PD-1 blocking antibody (R&D Systems/Techne, #AF1086) was included at the 10 μ g/ml.

Techniques: Staining, Expressing

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Expression and T cell Regulatory Action of the PD-1 Immune Checkpoint in the Ovary and Fallopian Tube

doi: 10.1111/aji.13649

Figure Lengend Snippet: ELISA assays were used to determine whether sPD and/or PD-1 ligands are present in HFF. PD-1 was detected in 20 of 32 samples measured (a, inset plot shows same data points between 0 and 1750 pg/ml expanded vertically, note single sample with greater than 15,000 pg/ml PD-1), while PD-L1 (b) and PD-L2 (c) were detected in all samples. Subsequent analysis detected full-length and shorter variants of each protein as enriched in HFF exosomal fractions (see Fig. S6). Representative examples of granulosa cells (gc) in large antral follicles, including HCGC are shown in panels d-f for each protein; this expression pattern may represent membrane and soluble forms of the proteins. PD-L2 was also detected consistently in cell-free contents (*) of ovarian (g) and fallopian tube (h) lymphvascular channels. Note that in the example ovarian lymphvascular channel (g), white blood cells are negative for PD-L2 (arrow), while the cell-free vessel contents in the fixed tissue are positive. Also note that the contents of the fallopian tube channel (h, *) are positive for PD-L2 while the contents of the vein at top right are negative; tubal luminal epithelium is at bottom right. (i) Western blot analysis of full-length and soluble PD-1 in HFF and media conditioned by HCGC, soluble form(s) enhanced by immunoprecipitation (IP) indicated by asterisks. Contents of lanes as follows: Lane 1: MW Marker, 2: media conditioned by HCGC (“HCGC media”) for 24 hours, 3: HFF, 4: HCGC media, anti-PD-1 IP, 5: HCGC media, IP no-first antibody control, 6: HCGC media, unrelated first antibody control, 7: HFF, anti-PD-1 IP, 8: HFF, IP no-first antibody control, 9: HFF, unrelated first antibody control, 10: HFF, IP no-first antibody control. (j) Western blot detection of ligands PD-L1 and -L2 in HFF. A first immunoprecipitation (IP) step was required to detect the ligands (equal amounts of protein are loaded with and without IP).

Article Snippet: Where specified, PD-1 blocking antibody (R&D Systems/Techne, #AF1086) was included at the 10 μ g/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Membrane, Western Blot, Immunoprecipitation, Marker

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Expression and T cell Regulatory Action of the PD-1 Immune Checkpoint in the Ovary and Fallopian Tube

doi: 10.1111/aji.13649

Figure Lengend Snippet: As seen with premenopausal specimens, PD-1 expression was highest in sparse cells with the appearance of surveilling immune cells in the ovarian cortex (a, inset), but was low or absent in cells of the OSE (b) and sub-surface epithelial inclusions (c, *). PD-L1 was widely expressed throughout postmenopausal ovarian cortex (d-f), and unlike the premenopausal ovary, was detectable at comparable levels in cell layers at the surface of the ovary (red brackets, panels d, e; compare to Fig. 1j). PD-L1 was also detected in cells of the OSE (d,e) and in cells of epithelial inclusions (example in f, note lack of surface OSE in this area). PD-L2 was mostly absent from postmenopausal specimens (g-i). No first antibody control shown in panel j. Ex - extra-ovarian space.

Article Snippet: Where specified, PD-1 blocking antibody (R&D Systems/Techne, #AF1086) was included at the 10 μ g/ml.

Techniques: Expressing

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Expression and T cell Regulatory Action of the PD-1 Immune Checkpoint in the Ovary and Fallopian Tube

doi: 10.1111/aji.13649

Figure Lengend Snippet: T cells were incubated with hFF without (a) or with (b) concurrent CD3/CD28 activation and IFNγ was measured 24 hours later. Critically, IFNγ was not detectable in any hFF sample prior to addition to T cells (not shown). In non-activated T cells, some hFF samples stimulated IFNγ production above baseline (blue points within oval; mean values for 3 replicates shown). When the same hFF samples were applied to CD3/CD28-activated T cells, net IFNγ production was calculated by subtracting the mean amount produced by vehicle-treated, activated controls (set to zero, dashed line). Compared to vehicle, IFNγ production was either diminished (“Inhibitory”) or enhanced (“Stimulatory”) by hFF treatment. All 5 samples shown to stimulate IFNγ production in non-activated T cells (a) were found to have “Stimulatory” action upon activated T cells (b). Panel c shows western blots of T cell lysates after the described treatments, probed for “active” P-Y248 PD-1 and β-actin housekeeping protein. hFF treatment corresponded to increased P-Y248 PD-1 in both Unactivated and Activated T cells, with a volume-dependent dose-response apparent in Activated T cells.

Article Snippet: Where specified, PD-1 blocking antibody (R&D Systems/Techne, #AF1086) was included at the 10 μ g/ml.

Techniques: Incubation, Activation Assay, Produced, Western Blot

Journal: American journal of reproductive immunology (New York, N.Y. : 1989)

Article Title: Expression and T cell Regulatory Action of the PD-1 Immune Checkpoint in the Ovary and Fallopian Tube

doi: 10.1111/aji.13649

Figure Lengend Snippet:

Article Snippet: Where specified, PD-1 blocking antibody (R&D Systems/Techne, #AF1086) was included at the 10 μ g/ml.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: Protein levels of exosomes and RFVs of PD-1+ or PD-L1+ exosomes in plasma of patients with HNSCC. A. protein concentrations of exosomes (μg/ml plasma) are significantly elevated in patients with active disease (AD) relative to those with no evidence of disease after therapy (NED); B. RFVs for PD-L1+ exosomes is significantly higher in AD patients, patients with positive lymph nodes and patients with the high (III/IV) UICC stage tumors at diagnosis. * p< 0.05; *** p< 0.0008. C. RFVs for PD-1 exosomes in plasma are not statistically different in HNSCC patients stratified by disease activity, lymph node or UICC status. The data presented as box plots are normalized to the frequency of exosomes/1mL of patients’ plasma in this and the subsequent figures.

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques: Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: RFVs for PD-L1+ and PD-1+ exosomes/mL plasma and levels of sPD-L1 in plasma of HNSCC patients. A. No correlation between RFVs for PD-L1+ and PD-1+ exosomes in plasma of HNSCC patients was observed: Spearman’s correlation at p=0.7, r=0.05. B. No correlation between RFVs for PD-L1+ exosomes in patients’ plasma and soluble PD-L1; Spearman’s correlation at p=0.85, r=0.03.

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Clinical significance of PD-L1 + exosomes in plasma of Head and Neck Cancer patients

doi: 10.1158/1078-0432.CCR-17-2664

Figure Lengend Snippet: CD69 and PD-1 expression on activated CD8+ T cells and down-regulation of CD69 expression by PD-L1high but not by PD-L1low exosomes. A. Representative flow cytometry showing expression levels of CD69 or PD-1 on the surface of activated CD8+ T cells. Decreasing levels of CD8+CD69+ on the surface of T cells co-incubated with PD-L1high exosomes. B, Downregulation of CD69 expression (% and MFI) on CD8+ T-cells after co-incubation with PD-L1high exosomes. In C, blocking of exosome-mediated down-regulation of CD69 expression on CD8+ T cells by anti-PD-1 Ab. Note that inhibition mediated by PD-L1high exosomes was almost completely reversed (% and MFI) by adding the PD-1 inhibitor. * p< 0.05; ** p<0.005

Article Snippet: Next, anti-PD-1 Ab, #AF1086 lot #ICA02 (purchased from R&D Systems) was used to block effects of exosome-mediated suppression.

Techniques: Expressing, Flow Cytometry, Incubation, Blocking Assay, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Lymphoid tissue fibrosis is associated with impaired vaccine responses

doi: 10.1172/JCI97377

Figure Lengend Snippet: This figure shows LN analysis from 3 different participants before vaccination and again at week 2 after vaccination. Participant 1996 has recognizable follicles before vaccination and formation of follicles after vaccination. The accumulation of PD1 staining cells within the secondary follicle (D) with green and blue staining cells in the secondary follicle (staining yellow) show an expected reaction to vaccination. Panel B shows participant 1682 with fewer, more poorly formed follicles at baseline and a lack of recognizable secondary follicles with vaccination. PD1 staining cells are not inside of the follicle structure. Participant 1688 has no recognizable follicles prior to vaccination and no response to vaccination. Scale bar indicates 20 μm.

Article Snippet: Slides were then washed 3 times in PBS for a total of 60 minutes and stained for 2 hours at RT with titrated amounts of the following conjugated antibodies: PD1-Alx488 (catalog FAB7115G; R&D Systems), CD20-efluor615 (clone L26; eBioscience), Ki67-Alx647 (clone B56; BD Biosciences), CD57-Alx680 (clone NK-1; in-house conjugated).

Techniques: Staining