Journal: bioRxiv

Article Title: Pre-Germinal Center Interactions with T Cells are Natural Checkpoints to Limit Autoimmune B Cell Responses

doi: 10.1101/2022.07.20.500862

Figure Lengend Snippet: Antigen-specific RFP + T and GFP + B cells were isolated and transferred to SMARTA recipient mice. Recipient mice were immunized 2d later with NPOVA, MOG tag , or NPMOG tag in CFA, as indicated. Intravital microscopy of draining popliteal lymph nodes was performed 2d post immunization. ( A ) Still frames from representative 3D videos showing a typical interaction between T (open arrow) and B cells (closed arrow) responding to NPOVA (top) or MOG tag (bottom) immunization. Note that the T/B interaction in the NPOVA system initiated prior to the beginning of the video, as was common for this model. The interaction was observed for a further ~35min before they separated (see Movie 3). The MOG T/B interaction shown is representative of the most common short interactions in this model system lasting less than 10min (see Movie 4). ( B ) Interactions were identified manually and their duration determined by identifying the timepoint when the cells first made contact or came into view, and then when they separated or left the field of view. ( C ) Interaction durations were binned between short (<5 min), medium (5-15 min), and long (>15 min). ( D ) Membrane entanglement was determined by identifying a frame with greatest contact between the interacting T and B cell. ( i ) The length of the membrane contact between the T and B cells was determined and expressed as ratio of the full T cell peripheral measurement ( ii ). Each symbol represents an individual interaction with data pooled from 4 independent experiments for each Ag group. P <0.05, **p <0.01, ***p <0.001, based on an ANOVA followed by a Students t test with Bonferroni correction was used for multiple comparisons.

Article Snippet: C57Bl/6, 2D2 TCR-transgenic , SMARTA TCR-transgenic (4694; Tg(TcrLCMV)327Sdz/JDvsJ), and OTII TCR-transgenic mice (4194; Tg(TcraTcrb)425Cbn/J) were purchased from Jackson Laboratories.

Techniques: Isolation, Intravital Microscopy, Membrane

Journal: bioRxiv

Article Title: Pre-Germinal Center Interactions with T Cells are Natural Checkpoints to Limit Autoimmune B Cell Responses

doi: 10.1101/2022.07.20.500862

Figure Lengend Snippet: Ag-sp. RFP + T and GFP + B cells were isolated and transferred to SMARTA recipient mice, which were immunized subcutaneously 2d later with NPOVA, MOG tag , or NPMOG tag in CFA, as indicated. Cells from draining inguinal lymph nodes were harvested 2d later and analyzed by FACS. ( A ) Responding Ag-sp. T cells were assessed for expression of ( i ) ICOS, ( ii ) CD11a, and ( iii ) CD150. Relative expression based on Geometric Mean Fluorescence Intensity compared to endogenous, naïve T cells from the same mouse is shown, with the value of 1 representing naïve expression levels. ( B ) Responding antigen-specific B cells were assessed for the expression of the corresponding receptors ( i ) ICOSL, ( ii ) ICAM-1, and ( iii ) CD150. B cell expression of ( iv ) MHC class II and ( v ) CD40 was also assessed. Relative expression compared to endogenous, naive B cells from the same mouse is shown, with the value of 1 representing naïve expression levels. ( C ) Relative IgD expression levels by all transferred GFP + B cells compared to endogenous GFP - B cells. Results pooled from at least 2 independent experiments for each marker, except for MHC II and CD40 (B iv and v ). Note that not every experiment stained for each marker, resulting in different numbers of points per plot. Each symbol represents an individual mouse. *p <0.05, **p <0.01, ***p <0.001, **** p <0.0001, based on an ANOVA followed by a Students t test with Bonferroni correction was used for multiple comparisons.

Article Snippet: C57Bl/6, 2D2 TCR-transgenic , SMARTA TCR-transgenic (4694; Tg(TcrLCMV)327Sdz/JDvsJ), and OTII TCR-transgenic mice (4194; Tg(TcraTcrb)425Cbn/J) were purchased from Jackson Laboratories.

Techniques: Isolation, Expressing, Fluorescence, Marker, Staining

Journal: Immunity

Article Title: Epithelial colonization by gut dendritic cells promotes their functional diversification

doi: 10.1016/j.immuni.2021.11.008

Figure Lengend Snippet:

Article Snippet: Mouse: OT-II RAG2-KO THY1.1 BL/6N , O. Lantz (Institut Curie) , N/A.

Techniques: Flow Cytometry, Blocking Assay, Purification, Recombinant, Staining, Gene Expression, Software

Journal: Experimental Hematology & Oncology

Article Title: CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

doi: 10.1186/s40164-022-00279-w

Figure Lengend Snippet: NI-1701 induces tumor cell phagocytosis by bone-marrow-derived dendritic cells and promotes cross-priming of CD8 + T cells. Bone marrow-derived mouse dendritic cells (CD11c + ) from BALB/c mice were cocultured for 2 h 30 ( a ) or 24 h ( b ) with Raji GFP hi tumor cells (1:1 ratio) in the presence of hIgG1 or NI-1701. Representative flow cytometry plots for hIgG1 and NI-1701 treated groups are depicted, and the percentage of phagocytosis is indicated (left panel). Phagocytic events were confirmed by imaging flow cytometry acquisition in the CD11c + GFP + gate with tumor cells in green fluorescence and CD11c + dendritic cells in red fluorescence (middle panel). The mean percentage of phagocytosis at 2 h 30 ± SD is shown (right panel), n = 5 independent experiments. b Percentage of residual Raji GFP hi tumor cells after 24 h of coculture with CD11c + DCs was determined by the analysis of remaining GFP + tumor cells within the total live cell gate. n = 5 experiments. c Bone-marrow derived dendritic cells from BALB/c mice were cocultured overnight with hemagglutinin-expressing Raji tumor cells (Raji HA-GFP) in the presence of human IgG1 or NI-1701 (ratio 1:1). The next day, HA-specific CD8 + T cells from CL-4 transgenic mice (bearing HA-specific TCR on CD8 + T cells) labelled with CellTrace violet were added (ratio 1:5). Analysis of CD8 + T cell proliferation was performed 3 days later. Plots represent an illustration of proliferating CD8 + T cells (left panel). Mean ± SD was calculated from 4 independent experiments (right panel). Significance was determined by unpaired t test. * p < 0.05, **** p < 0.0001

Article Snippet: CL-4 transgenic BALB/c mice with Hemagglutinin(HA)-specific TCR expressed by CD8 + T cells [ ] were kindly provided by Dr. Roland Liblau (Research Center Toulouse Purpan, CPTP-INSERM).

Techniques: Derivative Assay, Flow Cytometry, Imaging, Fluorescence, Expressing, Transgenic Assay

Journal: Blood

Article Title: B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance

doi: 10.1182/blood-2010-01-265975

Figure Lengend Snippet: Enhanced expansion of Ag-reactive CD8+ T cells by blockade of B7-H1/CD80 interaction. B6 mice were transferred intravenously with OTA-specific CD8 (OT-I) T cells and injected intravenously with 0.5 mg of OVA257-264 peptide. On day of peptide injection and 3 days later, the mice were treated intraperitoneally with 200 μg of 43H12 or control rat IgG. (A) PBMCs were harvested at the indicated time points, and a percentage of OT-I T cells in total CD8-positive cells was assessed in the 43H12-treated (●) or control IgG-treated (○) mice by flow cytometry. The data are shown as mean ± SEM. (B) Mice were given 100 μg of BrdU intraperitoneally on day 2 (top panels) or day 4 (bottom panels) after OVA peptide injection. Twenty-four hours after BrdU injection, spleen cells were harvested and BrdU incorporation in CD8/OVA-tetramer double-positive OT-I T cells was analyzed by flow cytometry (black histogram). As background level, OT-I T cells in the mice without BrdU administration were stained similarly (gray histogram). (C) Spleen cells were harvested 4 days after OVA peptide injection and annexin V staining in CD8/OVA-tetramer double-positive OT-I T cells was analyzed by flow cytometry (black histogram). Background level without annexin V staining is also shown (gray histogram). All experiments were independently repeated at least 3 times, and the representative data are shown. The numbers in the histogram indicate the percentage of positively stained cells.

Article Snippet: OTA-specific CD8 (OT-I) T-cell receptor (TCR)–transgenic mice were purchased from Taconic.

Techniques: Injection, Control, Flow Cytometry, BrdU Incorporation Assay, Staining

Journal: Blood

Article Title: B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance

doi: 10.1182/blood-2010-01-265975

Figure Lengend Snippet: A role of T cell–associated CD80 in the inhibitory effects of B7-H1/CD80 interaction. (A) WT B6 mice or CD80-KO mice were transferred intravenously with OT-I T cells. In panel B, B6 mice were transferred intravenously with WT, B7-H1-KO, or CD80-KO background OT-I T cells. In both settings, the recipient mice were injected intravenously with 0.5 mg of OVA257-264 peptide, and treated intraperitoneally with 200 μg of 43H12 or control rat IgG on day of peptide injection and 3 days later. Splenocytes were harvested 5 days after peptide injection, and the percentage of OT-I T cells in CD8-positive population was assessed by flow cytometry. (C) B6 mice were transferred intravenously with OT-I T cells and injected intravenously with 0.5 mg of OVA257-264 peptide. On day 3 and day 5, CD8/OVA-tetramer double-positive OT-I T cells was stained with anti-CD80 mAb and analyzed by flow cytometry (gray histogram). Nonstained background levels of the same cells are also shown (open histogram). All experiments were repeated at least 3 times, and the representative data are shown. The numbers in the histogram indicate the percentage of positively stained cells.

Article Snippet: OTA-specific CD8 (OT-I) T-cell receptor (TCR)–transgenic mice were purchased from Taconic.

Techniques: Injection, Control, Flow Cytometry, Staining

Journal: Blood

Article Title: B7-H1/CD80 interaction is required for the induction and maintenance of peripheral T-cell tolerance

doi: 10.1182/blood-2010-01-265975

Figure Lengend Snippet: Prevention and restoration of CD8+ T-cell anergy by blockade of B7-H1/CD80 interaction. B6 mice were transferred intravenously with OT-I T cells and injected intravenously with 0.5 mg of OVA257-264 peptide. (A) On day of peptide injection and 3 days later, the mice were treated intraperitoneally with 200 μg of 43H12 (●) or control rat IgG (○). Thirty-four days after initial peptide injection, the mice were rechallenged intravenously with 0.5 mg of OVA257-264 peptide, and percentages of CD8/OVA-tetramer double-positive OT-I T cells in PBMCs were assessed by flow cytometry at the indicated time points. Fold expansion of OT-I T cells was calculated by dividing OT-I T-cell percentages after rechallenge by that before rechallenge in individual mice. (B) Twenty days after the initial OVA peptide injection, the mice were rechallenged with 0.5 mg of OVA257-264 peptide and treated intraperitoneally with 200 μg of 43H12 (●) or control rat IgG (○) on day of peptide rechallenge and 3 days later. Fold expansion of OT-I T cells in PBMC was assessed as in panel A at the indicated time points. (C) Twenty days after the initial OVA peptide injection, the mice were left untreated (left panel) or rechallenged with 0.5 mg of OVA257-264 peptide (right panel). Twenty-four hours later, CD8/OVA-tetramer double-positive OT-I T cells in the spleen were stained with anti-CD80 mAb and analyzed by flow cytometry (gray histogram). Nonstained background levels of the same cells are also shown (open histogram). All experiments were repeated at least 3 times and the representative data are shown. The numbers in the histogram indicate the percentage of positively stained cells.

Article Snippet: OTA-specific CD8 (OT-I) T-cell receptor (TCR)–transgenic mice were purchased from Taconic.

Techniques: Injection, Control, Flow Cytometry, Staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: B Cell Depletion With an Anti-CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

doi: 10.1111/ajt.13483

Figure Lengend Snippet: OT1 mice were injected with 5D2 IgG2a anti-CD20 antibody (250 µg given intraperitoneally). The percentages of B cells among total lymphocytes were evaluated at different time points in the peripheral blood and bone marrow (BM) (A) as well as lymph nodes (LN) and spleen (B) of anti-CD20 mAb-treated (solid symbols) or control untreated (open symbols) mice via fluorescence activated cell sorter staining with an anti-CD19 antibody. The percentages of CD19+ B cells among lymphocytes of control untreated OT1 mice were as follows: bone marrow: 52%, peripheral blood: 48%, lymph nodes: 28%, and spleen: 46%. The results are representative of three to four mice tested at each time point.

Article Snippet: BALB/c (K d A d E d L d D d ), C3H (K k A k E k L k D k ), C57BL/6 (K b A b E − L b D b ), anti-OVA TCR transgenic OT1 mice (recognize MHC class I K b + OVA peptide 254–267, SIINFEKL) and transgenic Act mOVA mice were obtained from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Injection, Control, Fluorescence, Staining

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: B Cell Depletion With an Anti-CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

doi: 10.1111/ajt.13483

Figure Lengend Snippet: The frequencies of γ-interferon (γ-IFN)-producing memory T cells activated through allorecognition were investigated 40 days after transplantation of OT1(A) or BALB/c (B) mice with ActmOVAorB6skin grafts, respectively. Panel (C) shows the memory responses of T cells collected from µMT B cell deficient mice transplanted with a BALB/c skin graft. Black bars correspond to anti-CD20 mAb-treated recipients while gray bars show T cell responses measured with control untreated mice.

Article Snippet: BALB/c (K d A d E d L d D d ), C3H (K k A k E k L k D k ), C57BL/6 (K b A b E − L b D b ), anti-OVA TCR transgenic OT1 mice (recognize MHC class I K b + OVA peptide 254–267, SIINFEKL) and transgenic Act mOVA mice were obtained from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Transplantation Assay, Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: B Cell Depletion With an Anti-CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

doi: 10.1111/ajt.13483

Figure Lengend Snippet: (A) OT1 mice received two skin grafts from Act mOVA donors at day 0 and day 100. Five days before the second graft, some recipients were treated with anti-CD20 mAbs. (B) This panel shows the frequencies of alloreactive memory T cells producing γ-interferon (γ-IFN) in untreated (gray bars) or mice treated with anti-CD20 mAbs (black bars) measured 40 days after the second graft via in vitro stimulation with intact donor OVA transgenic antigen-presenting cells (APCs) (endogenous processing) or recipient B6 APCs + OVA-transgenic donor sonicates (exogenous processing). Controls included T cells stimulated with syngeneic APCs, syngeneic sonicates, or medium. The results are expressed as numbers of γ-IFN spots per million T cells and represent three mice tested individually±standard deviation. Panel (C) shows the percentages of first (dotted lines) and second graft (solid lines) survival overtime for untreated OT1 control mice (gray lines) and anti-CD20 mAb-treated mice (black bars).

Article Snippet: BALB/c (K d A d E d L d D d ), C3H (K k A k E k L k D k ), C57BL/6 (K b A b E − L b D b ), anti-OVA TCR transgenic OT1 mice (recognize MHC class I K b + OVA peptide 254–267, SIINFEKL) and transgenic Act mOVA mice were obtained from the Jackson Laboratory (Bar Harbor, ME).

Techniques: In Vitro, Transgenic Assay, Standard Deviation, Control