Journal: Mucosal immunology

Article Title: Age-associated Antigen-Presenting Cell Alterations Promote Dry-Eye Inducing Th1 cells

doi: 10.1038/s41385-018-0127-z

Figure Lengend Snippet: A. FITC-conjugated OVA peptide (OVAp) was topically applied to the ocular surface of young and aged female mice. Uptake of the fluorescent peptide was evaluated by confocal microscopy thirty minutes after application. Laser confocal microscopy of whole-mount conjunctiva stained for CD11b (A) and CD11c (B) thirty minutes after placing FITC-conjugated OVAp antigen on the ocular surface; Z-stack option from the epithelium (e) to stroma (s) showing the distribution in immune cells and OVAp. Goblet cell associated passages (columns) were easily identified (asterisks). Frequent OVA + CD11b + and OVA + CD11c + cells were seen in aged eyes (arrows). Squares in z-stacks are magnified on the right. E = epithelium, s = stroma. n = two animals/age. B-C. Water or FITC conjugated OVAp was applied to the ocular surface at 0 and 12 hours. Right, and left conjunctivas (CJ) were harvested and pooled, and cervical lymph nodes (CLN) were collected and prepared for flow cytometry 24 hours later the first OVAp administration (total of two applications). CD11c + cells were then plotted versus MHC II and further gated into CD103 + or CD11b + cells. MHC II + CD11c− cells were identified and plotted versus F4/80 and CD11b to gate MHC II + CD11b + F4/80 + cells. Median fluorescence intensity (MFI) of CD86 staining was then analyzed in these populations. Dendritic cells were MHC II + CD11c + CD11b + and macrophages were MHC II + CD11c−CD11b + F4/80 + . The frequency of OVAp + cells was then determined in each population. B. Accumulative data of flow cytometry analysis. C. Representative dot plots of OVAp laden APCs in the conjunctiva and CLN. Means ± SEM, n = four to seven mice/age; biological replicates from two independent experiments were averaged. D. Laser scanning confocal microscopy of whole mount corneas and bulbar conjunctivas stained for occludin (green) with Hoechst 33342 (blue) nuclear counterstaining (n = 3). Arrows in the cornea panel indicate desquamating cell and arrowheads indicate loss of occludin immunoreactivity in between two cells. W = week, M = month, NS = non-significant, *P<0.05; **P<0.01, ***P<0.001 Mann Whitney U test age comparison.

Article Snippet: A topical dose of 2.5 μl of FITC labeled OVA 323–339 peptide (OVAp, 5 mg/mL, KareBay Biochem Inc, Monmouth Junction, NJ) was topically instilled in each eye of young and aged mice at times 0 and 12 hours and the animals were euthanized 12 hours after the second dose (24 hours total) and compared to mice that received vehicle dosing (water).

Techniques: Confocal Microscopy, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY, Comparison

Journal: The Journal of Clinical Investigation

Article Title: A narrow T cell receptor repertoire instructs thymic differentiation of MHC class Ib–restricted CD8 + regulatory T cells

doi: 10.1172/JCI170512

Figure Lengend Snippet: ( A ) WT B6 mice were immunized with Kb –/– Db –/– DC loaded with FL9 peptide on days 0, 8, and 15. At day 22, Qa-1–FL9–specific CD8 T cells were detected by tets (Qa-1–FL9–PE and Qa-1–FL9–APC) within CD44 + CD122 + Ly49 + CD8 T cells. ( B ) TCR repertoire of Qa-1–FL9 Tet + CD8 T cells. Single Qa-1–FL9–PE + Qa-1–FL9–APC + cells were sorted and subjected to sequencing for TCRα and TCRβ. Thirty-nine of TCRα and TCRβ pairs were analyzed based on their TCR V gene segments. Relative usage of TCRα and TCRβ V genes by these Tet + single cells is depicted by donut charts. ( C ) TCR repertoire of Qa-1–Hsp60 Tet + CD8 T cells. Single Qa-1–Hsp60–PE + Qa-1–Hsp60–APC + cells were sorted and sequenced for TCRα and TCRβ. Relative usage of TCRα and TCRβ V genes by these Tet + single cells is depicted by donut chart. ( D ) Frequency and phenotype of Vα3.2 + Vβ5 + cells within Ly49 + CD8 cells in the spleens and LNs of WT B6, Qa-1.D227K–KI and Qa-1–KO mice at 8 weeks of age ( n = 6/group). ( E ) Qa-1–dependent differentiation of FL9 T cells: tet-mediated detection of TCR in 58C hybridoma transduced with FL9.2 and FL9.8 TCR (upper panel). Responsiveness of FL9.2-TCR– and FL9.8-TCR–expressing hybridoma upon stimulation with increasing dose of peptides measured by CD69 expression (lower panel). ( F ) Measurement of Qa-1–FL9 binding affinity of FL9.2 and FL9.8 TCR. FL9.2 TCR + and FL9.8 TCR + hybridoma were labelled with Qa-1–FL9–PE tets and incubated in the presence of anti-Qa-1 antibodies for the indicated time. Percentages of PE + cells were measured at different time points as a measurement of tet dissociation level. ( G ) Tg TCR + cells in TCR + thymocytes and percent of active-Caspase–3 + PD1 + cells in DP (CD4 + CD8 + ) thymocytes in OT-I → WT B6, FL9.2 Tg → WT B6 BM chimera 8 weeks after BM reconstitution. ( H ) Ki67 and CD44 expression by OT-I and FL9.2 TCR Tg CD8 + T cells was measured as an indication of Ag encounter in the spleen and liver of OT-I → WT B6 and FL9.2 Tg → WT B6 BM chimera 8 weeks after BM reconstitution. OT-1 is also used as a control in . * P < 0.05, ** P < 0.01, *** P < 0.001, according to Wilcoxon-Mann-Whitney rank sum test.

Article Snippet: I-A b /Ova 323-339 tets were purchased from MBL International.

Techniques: Sequencing, Transduction, Expressing, Binding Assay, Incubation, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: A narrow T cell receptor repertoire instructs thymic differentiation of MHC class Ib–restricted CD8 + regulatory T cells

doi: 10.1172/JCI170512

Figure Lengend Snippet: ( A ) in vitro, conA-stimulated CD4 cells from WT B6, Qa-1.D227K–KI, KbDb-KO and ERAAP-KO mice were cocultured with FL9.2 T cells isolated from FL9.2 TCR Tg mice. WT CD4 cells loaded with the FL9 peptide were used as a positive control. After 20 hours, CD69 expression on FL9 Tg T cells were measured as a readout of TCR stimulation. ( B ) In vivo, WT or D227K mice were immunized with OT-II peptides in CFA. After 7 days, CD4 (CD4 + CD25 – ) cells were isolated from immunized mice and transferred into WT B6 hosts with or without FL9 TCR Tg T cells followed by immunization with OT-II/CFA. Detection of I-A b /Ova 323-339 -specific CD4 T cells in the spleen of hosts by I-A b /Ova 323-339 tets (upper). Percent and numbers of I-A b /Ova 323-339 tet + (upper) and I-A b /Ova 323-339 tet – activated (lower) CD4 cells recovered from adoptive hosts (middle and right). ( C ) Qa-1 expression by I-A b /Ova 323-339 tet + and tet – CD4 cells. ( D ) WT B6 and D227K mice were immunized with Ova/CFA and injected with isotype or anti-Vα3.2 antibodies on day 0, before boosting on day 8 with Ova/IFA along with antibody injection. Frequency of I-Ab/Ova 323-339 tet + CD4 cells in blood were assessed on day 15. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: I-A b /Ova 323-339 tets were purchased from MBL International.

Techniques: In Vitro, Isolation, Positive Control, Expressing, In Vivo, Injection