np ova  (Biosearch Technologies Inc)

Journal: STAR Protocols

Article Title: Protocol for an in vivo CRISPR screen for germinal center B cells in mice using ecotropic retrovirus

doi: 10.1016/j.xpro.2026.104586

Figure Lengend Snippet: Immunization experiment scheme for in vivo screen Host mice (IOMA gl) were immunized with OVA in alum. At week 2 after immunization, B cells from B1-8 hi , LSL-Cas9, AID-Cre, Kappa KO animals were transduced with pooled sgRNA library retrovirus and adoptively transferred to the hosts. Subsequently, the host animals were boosted with NP-OVA in alum at day 0 and 2 and administered with anti-DEC205-OVA mAb at day 6.5. Spleens were harvested and processed for cell sorting at day 10.

Article Snippet: NP-OVA (conjugation ratio: 21) , Biosearch Technologies , cat# N-5051.

Techniques: In Vivo, Transduction, FACS

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Breeding scheme for generating CNS1-YFP-RFP OT-II Tg mice. Foxp3 ΔCNS1-Cre-Thy1.1 (expressing Cre under the Foxp3 promoter with CNS1 deleted) were crossed with Rosa26 loxP-stop-loxP-YFP and Foxp3 IRES-RFP mice. Cre-mediated excision of the loxP-flanked stop cassette at the Rosa26 locus enables YFP expression, while RFP reports Foxp3 expression. CNS1-YFP-RFP mice were further crossed with OT-II TCR transgenic mice (specific for OVA 323–339 ). ( B ) Experimental setup for adoptive transfer and immunization. CD45.1 + CD45.2 + CD4 + YFP + RFP + OT-II Tg tT reg cells were transferred intravenously into CD45.2 + recipients. One day later, mice were immunized intraperitoneally with PBS or NP-OVA plus alum. Spleens were analyzed by flow cytometry on day 7. ( C ) TCR stimulation influences tT reg cells stability. (left) Flow cytometry plots showing Thy1.1 and Foxp3 expression in CD4 + YFP + tT reg cells from PBS (n=4) or NP-OVA plus alum (n=9) immunized recipient spleens. Numbers indicate gate frequencies. (right) Quantification of ex-T reg cells (Foxp3 − Thy1.1 − , red box). ( D ) Altered peptide ligands (APLs) with varying TCR affinities. OVA-derived APLs are shown with OT-II TCR-binding regions highlighted: OVA 323–339 (red), OVA 327–339 (orange), OVA 328–339 (purple), and OVA 329–339 (blue). Affinity decreases sequentially. ( E ) Frequency of transferred OT-II Tg T reg cells in recipient mice. Proportion of CD45.1 + CD45.2 + OT-II Tg T reg cells among total CD4 + cells after immunization with indicated OVA peptides plus alum. ( F ) Ex-tT reg cells generation correlates with TCR signal strength. Frequency of Foxp3 − Thy1.1 − ex-tT reg cells among activated CD4 + CD44 + YFP + cells after peptide immunization. ( G ) Intense TCR signals drive T FR cells instability. Representative flow cytometry plots showing T FR cells differentiation and ex-tT reg cells generation following high-affinity TCR stimulation. ND means not displayed due to being below the detection limit. (E) to (G) show pooled data from two independent experiments with OVA 323–339 (n=7), OVA 327–339 (n=5), OVA 328–339 (n=4), and OVA 329–339 (n=4). All flow cytometry data were acquired from two independent experiments. Error bars show mean ± SD. P values were calculated using the unpaired Student’s t -test. ** P <0.01.

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Expressing, Transgenic Assay, Adoptive Transfer Assay, Flow Cytometry, Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Immunization and analysis scheme. CNS1-YFP-RFP mice received footpad injections of 10 μg NP-OVA plus alum. Draining popliteal lymph nodes (dpoLNs) and non-draining popliteal lymph nodes(ndpoLNs) were analyzed by flow cytometry for PD-1 and CXCR5 expression on CD4 + YFP + tT reg cells at indicated time points. ( B ) T FR differentiation kinetics. (left) Representative flow plots of PD-1 versus CXCR5 on CD4 + CD44 + YFP + tT reg cells. (right) Quantification of T FR cell frequency (top) and numbers (bottom) in dpoLNs versus ndpoLNs. Day4 (n=3), day7 (n=5), day10 (n=4), day14 (n=3), day21(n=2). Flow cytometry data were acquired from two independent experiments. ( C ) scRNA-seq experimental design. PD-1 + CXCR5 + YFP + T FR cells from immunized mice were FACS-sorted for single-cell RNA and TCR sequencing. ( D ) T FR cell subsets heterogeneity. UMAP projection of 7 transcriptionally distinct clusters. Dot plot shows relative expression of Foxp3, Pdcd1 (PD-1), and Cxcr5 (red: high; gray: low). ( E ) Developmental regulation of CD25. Il2ra (CD25) expression patterns in early-(C3/C5/C6) versus late-stage (C1/C2/C4) T FR cell clusters. ( F ) Ex-T reg cytokine production. Il21 expression map showing Foxp3 - Il21 + cells (purple) within late-stage T FR cell clusters. ( G ) TCR clonal analysis. Six expanded clonotypes (colored) with corresponding CDR3α/β sequences. ( H ) TCR signaling assay. Schematic of NFAT-GFP reporter system in TCR-transduced hybridomas cultured with APCs plus antigen/LPS. ( I ) Antigen specificity validation. GFP induction in OTREG-1 hybridomas stimulated with OVA peptide variants. Representative flow cytometry data of four independent experiment(left). Each data point represents one independent experiment. ( J ) Tetramer reagents. Designed OVA peptide sequences (OVA 302-316 , OVA 303-313 , OVA 304-314 ) for TCR specificity testing. ( K ) Tetramer binding confirmation. Flow cytometry analysis of TCRβ versus OVA-tetramer staining (control: hCLIP 87-101 tetramer).

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Flow Cytometry, Expressing, Single Cell, Sequencing, Cell Culture, Biomarker Discovery, Binding Assay, Staining, Control

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Kinetics of T FH cell differentiation post-immunization. (left) Flow cytometry plots showing PD-1 and CXCR5 expression on CD4 + CD44 + YFP - RFP - T FH cells in draining and non-draining popliteal lymph nodes (dpoLNs and ndpoLNs) at indicated time points. (right) Quantification of T FH cell frequency (top) and absolute cell numbers (bottom). Day4 (n=3), day7 (n=5), day10 (n=4), day14 (n=3), day21(n=2). Flow cytometry data were acquired from two independent experiments. ( B ) Transcriptional signatures of T FR clusters. Heatmap of differentially expressed genes (DEGs) across Tfr cell clusters (C1–C7). ( C ) Consensus gene signatures of late-stage T FR cells. Venn diagram comparing DEGs between: Late-stage (C1/C2/C4) versus early-stage (C3/C5/C6) T FR cell clusters; CD25 − versus CD25 + T FR cells ( D ) Antigen-specific TCR activation assay. GFP induction in hybridomas expressing six expanded TCR clonotypes (OTREG-1, TCR2–6) after 48 h co-culture with: NP-OVA plus APCs (positive conditions), NP-OVA without APCs and NP-BSA or medium (negative controls). Representative flow cytometry data of two independent experiment. ( E ) UMAP highlighting the dominant OTREG-1 TCR clone with Il21 expression.

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Cell Differentiation, Flow Cytometry, Expressing, Activation Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Predicted OVA peptide affinities. Structural modeling of six OVA-derived peptides ranked by estimated TCR-binding affinity. ( B ) Dose-response to OVA 302-316 peptide. Percentage of GFP + OTREG-1 (purple) and OT-II (orange) hybridoma cells after 48h coculture with indicated concentrations of OVA 302-316 peptide. Data represent mean ± SD of triplicate experiments. ( C ) Dose-response to OVA 323-339 peptide. Percentage of GFP + cells for each hybridoma line cultured with graded doses of OVA 323-339 peptide. Data represent mean ± SD of triplicate experiments. ( D ) Dose-response to NP-OVA. Percentage of GFP + OTREG-1 (purple) and OT-II (orange) hybridoma cells after 48 h coculture with indicated concentrations of NP-OVA. Data represent mean ± SD of triplicate experiments. ( E ) Dose-response to OVA 302-339 . Percentage of GFP + OTREG-1 (purple) and OT-II (orange) hybridoma cells after 48 h coculture with indicated concentrations of OVA 302-339 peptide. Representative flow cytometry data of four independent experiment(left). Error bars show mean ± SD, P values were calculated using multiple unpaired Student’s t -test, **** P <0.0001.

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Derivative Assay, Binding Assay, Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Altered thymocyte development in OTREG-1 Tg mice. (left) Flow cytometry plots of CD4 and CD8 expression in thymocytes from 4–5-week-old WT and OTREG-1 Tg mice. (right) Quantification of DN (double-negative), DP (double-positive), CD4 SP (single-positive), and CD8 SP populations. Data are pooled from all mice. Female WT(n=9), Male WT(n=10), Female OTREG-1 Tg (n=10), Male OTREG-1 Tg (n=10). ( B ) Impaired thymic maturation of OTREG-1 Tg T cells. (left) CD24 and Qa-2 expression on TCRβ hi CD4 SP thymocytes. (right) Frequency of mature CD24 lo Qa-2 hi cells among TCRβ hi CD4 SP thymocytes. Female WT(n=9), Male WT(n=10), Female OTREG-1 Tg (n=10), Male OTREG-1 Tg (n=10). ( C ) Homeostatic proliferation of OTREG-1 Tg T cells. (left) Histograms showing proliferation (CFSE dilution) of splenic OTREG-1 Tg (n=4) and OT-II Tg (n=4) CD4 + T cells. (right) Quantification of divided cells at day 4 and day 7. ( D ) Competitive proliferation assay design. Naive CD4 + CD25 − CD44 lo CD62 hi T cells from CD45.2 + OTREG-1 Tg and CD45.1 + OT-II Tg mice were mixed 1:1, CFSE-labeled, and transferred into CD45.1 + CD45.2 + WT hosts. Recipients were immunized i.p. with PBS (n=2) or NP-OVA plus alum (n=11); donor cells were analyzed 3 days later. ( E ) Activation of OTREG-1 versus OT-II Tg T cells. Flow cytometry plots of CFSE dilution (proliferation) and CD44 expression in donor CD45.2 + OTREG-1 and CD45.1 + OT-II Tg CD4 + T cells. ( F ) T FH differentiation potential. Flow cytometry plots of PD-1 and CXCR5 expression on donor OTREG-1 and OT-II Tg CD4 + T cells. ( G ) Quantification of activated/proliferating T cells. (upper) Frequency of CFSE lo CD44 hi cells among OTREG-1 (CD45.2 + ) and OT-II (CD45.1 + ) Tg CD4 + T cells. (lower) Frequency of PD-1 hi CXCR5 hi cells among OTREG-1 (CD45.2 + ) and OT-II (CD45.1 + ) Tg CD4 + T cells. All flow cytometry data were acquired from at least two independent experiments. Error bars show mean ± SD, P values were calculated using unpaired Student’s t -test, not significant(ns), * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Flow Cytometry, Expressing, Proliferation Assay, Labeling, Activation Assay

Journal: bioRxiv

Article Title: TCR signal strength determines T reg instability and discrimination of self versus non-self antigens

doi: 10.64898/2026.02.12.705537

Figure Lengend Snippet: ( A ) Experimental design for assessing the stability of OTREG-1 T reg cells. CD45.2 + CD4 + YFP + OTREG-1 Tg tT reg cells were sorted from CNS1-YFP-RFP OTREG-1 Tg mice and adoptively transferred into CD45.1 + WT recipients via tail vein injection. Mice were immunized i.p. with NP-OVA plus alum or PBS (control) the following day, and donor cells were analyzed by flow cytometry 7 days post-transfer. ( B ) Homeostasis of transferred tT reg cells. Frequency of donor OTREG-1 Tg tT reg cells (CD45.2 + ) among splenic CD4 + T cells in recipient mice. ( C ) Stability of transferred tT reg cells. Foxp3 and Thy1.1 expression in donor tT reg cells from recipient spleens. Numbers indicate gate frequencies. ( D ) T FR cells differentiation. PD-1 and CXCR5 expression on donor tT reg cells following immunization. The percentage of PD-1 hi CXCR5 hi Tfr cells is shown. ( E ) Foxp3 stability of T FR versus non-T FR cell populations. Foxp3 expression in OVA-specific PD-1 lo CXCR5 lo (non-T FR ) and PD-1 hi CXCR5 hi (T FR ) cells from immunized mice. ( F ) Antigen challenge protocol for OTREG-1 retrogenic mice. CNS1-YFP-RFP OTREG-1 retrogenic mice received NP-OVA plus alum immunizations at day 0 and day 14, with analysis performed 14 days after the second immunization. ( G ) Antigen-specific versus bystander activation. PD-1 and CXCR5 upregulation on NGFR + CD4 + Tconv cells and tT reg cells from retrogenic mice, comparing OVA-specific (NGFR + ) versus non-specific (NGFR - ) populations. ( H ) Foxp3 instability in T FR versus non-T FR cell subsets. (left) Flow cytometry analysis of Foxp3 expression in PD-1 lo CXCR5 lo and PD-1 hi CXCR5 hi cell populations gated on CD4 + CD45.1 + YFP + NGFR + cells. (right) Quantification of ex-tT reg cells (Foxp3 - Thy1.1 - ) in each subset. (B) to (E) shows pooled data from three independent experiments with PBS group (n=6) and NP-OVA group (n=11). (G) to (H) shows pooled data from three independent experiments (n=14). All flow cytometry data were acquired from at least two independent experiments. Error bars show mean ± SD, P values were calculated using paired (G to H) or unpaired (B to E) Student’s t -test, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: For footpad immunization, mice received 10 μg of alum-precipitated NP-OVA (Biosearch Technologies) as previously described ( ).

Techniques: Injection, Control, Flow Cytometry, Expressing, Activation Assay