Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: A technical control of non-stimulated (NST) PMBCs was included. a, b, c) Percentage CD4 T cells in PBMCs stimulated with S peptides in the H donors (a) and in O patients (b). c) Comparison between H donors and O patients. d, e, f) Percentage CD4 T cells in PBMCs stimulated with M peptides in H donors (d) and in O patients (e). f) Comparison between H donors and O patients. g, h, i) Percentage CD4 T cells in PBMCs stimulated with N peptides in the H donors (g) and in O patients (h). i) Comparison between H donors and O patients. a-i) Krustal-Wallis test followed by Dunn’s test for pair-wise comparisons was employed. j) Dot plot of the percentage of S and M-specific CD4 T cells. The U of Mann-Whitney was used to test differences. k) Dot plot of S- and M-specific CD4 T cells in vaccinated H donors, from samples collected at the indicated timelines. Pair-wise comparisons were performed using the U of Mann-Whitney. l, m) Relative percentages of CD4 T cell differentiation phenotypes in H donors (l) and O patients (m) . N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L- CD45RA-) and effector (CD62L-CD45RA+) phenotypes. Relevant statistical comparisons are detailed in . *, **, ***, **** indicate P <0.05, <0.01, <0.001 and <0.0001, respectively.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Control, Comparison, MANN-WHITNEY, Cell Differentiation

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: Representative flow cytometry density plots of CD154 and CD137 co-expression profiles in CD4 T cells from donors before and after stimulation with S-peptides, as indicated.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with CD62L-CD45RA co-expression profiles in CD4 T cells before and after the stimulation with S-peptides. Quadrants were established with unstained controls. Percentages of the corresponding populations are shown within the quadrants. b , c) CD4 T cell phenotypic changes in H-V (b) and H-CoV-V (c) donors. (-) and (+S), non-stimulated and S-peptide stimulation. N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L- CD45RA-) and effector (CD62L-CD45RA+) phenotypes. d) Phenotypic changes in CD4 T cells within O-CoV-V before and after stimulation with S-peptides. e , f) Effects of previous CoV infection in vaccinated H donors and O patients over T cell phenotypes after stimulation with S-peptides. b-f) Relevant statistical comparisons are indicated by ANOVA followed by pair-wise comparisons with Tukey’s test. g , h) Relative percentages of CD4 T cell differentiation phenotypes in H-V and O-V (g) and in H-CoV-V and O-CoV-V (h) CD27+ CD28+, CD27-CD28+ and CD27+ CD28+ indicate poorly differentiated, intermediate differentiated and highly differentiated T cell phenotypes. U of Mann-Whitney was used to test for significance.*, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing, Infection, Cell Differentiation, MANN-WHITNEY

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with the expression of INFγ and IL-17 before and after stimulation with S peptides in CD4 T cells. b) In CD8 T cells.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a, b, c) Percentage of IFNγ-CD4 T cells specific for the S protein in H and O groups. d, e, f) Percentage of IFNγ-CD4 T cells specific for the M protein in H and O groups. g, h, i) Percentage of IL17-CD4 T cells specific for the S protein in H and O groups. j, k, l) Percentage of IL17-CD4 T cells specific for the M protein in H and O groups. a-l) Statistical significance was evaluated with Kruskal-Wallis followed by Dunn’s pair-wise comparisons. m, n) Percentage of CD4 T cells expressing IL-17 and IFNγ expression in H-V donors that completed the vaccine regime before sample collection in the indicated timelines, for both S and M proteins. Significance was tested with the U of Mann-Whitney test. NST, technical control of non-stimulated PMBCs. *, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Expressing, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Flow cytometry contour plots demonstrating the loss of the endogenous splenic CD4 + Foxp3 -GFP + Treg population in Foxp3 GFP-DTR mice following administration of diphtheria toxin (DTx). (C) Lung and spleen Foxp3 -GFP + Treg cell quantification in Foxp3 GFP-DTR mice that received influenza A virus but no DTx (No DTx + flu, n = 3) or influenza and DTx (DTx + flu, n = 4) at 13 DPI compared with mice that received DTx but no influenza (DTx No flu, n = 3). (D) Mass over time of Foxp3 GFP-DTR mice receiving no DTx or DTx until pre-specified timepoints (DTx withdrawal) following intra-tracheal inoculation of 5 plaque-forming units (PFU) of influenza A/WSN/33 H1N1 virus (n = 5 per group). (E) Representative lung histopathology (H&E staining) at 60 DPI of control (No DTx) and DTx withdrawal mice following influenza A virus infection. Original magnification x10, scale bar = 1 mm. (F) Metagene analysis of DNA methylation across Treg-SE of Tregs recovered from the mice described in 1D compared with splenic nTregs and iTregs from culture. Data presented as mean and SD. * q < 0.05 according to one-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C).* q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (D).

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Virus, Histopathology, Staining, Control, Infection, DNA Methylation Assay

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Flow cytometry contour plots for phenotyping of the CD4 + Foxp3 -GFP + tdTomato + iTreg population following culture in the presence of αCD3/αCD28, IL-2, TGF-β, and tamoxifen. (B) Mass over time of Foxp3 GFP-DTR mice receiving adoptive transfer of nTreg (n = 27), PBS (n = 21), Tconv (n = 25), or iTreg (n = 18) cells 5 days following intra-tracheal inoculation of 6.5 PFU of influenza A/WSN/33 H1N1 virus. Negative controls included mice that received influenza but no DTx (no DTx + flu, n = 9) and DTx but no influenza (DTx no flu, n = 3). (C) Total number of lung CD45 + cells at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10). (D-H) Total number of lung CD326 + CD31 − CD45 − cells (D), CD326 + MHCII + T1A − cells (E), KRT5 + CD326 + cells (F), Ki-67 + CD326 + MHCII + cells (G), and CD326 − CD31 + cells (H) at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10) cells. * q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (B). Data presented as mean and SD (C, F-H) with * q < 0.05 according to multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C). UHRF1 is dispensable for iTreg FOXP3 induction and stability but is required to maintain transcriptional and epigenetic programs in vitro .

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Adoptive Transfer Assay, Virus, MANN-WHITNEY, In Vitro

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Frequency of Foxp3 -GFP + and Foxp3 -GFP − tdTomato + (ex-FOXP3) cells in culture of iTregs with UHRF1 deleted concurrent with (early) or 5 days after (delayed) FOXP3 induction compared with Uhrf1 +/+ controls on days 5 and 12 of culture (n = 3 per group). (C) Division index of CD4 + CTV + Foxp3 -GFP − splenic responder T cells co-cultured for 72 hours with varying ratios of Uhrf1 +/+ CD4 + Foxp3 -GFP + iTregs, Uhrf1 fl/fl CD4 + Foxp3 -GFP + iTregs, or Uhrf1 +/+ nTregs (n = 3 per group). (D) Principal component analysis of 6,978 differentially expressed genes from sorted cells described in A, identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (E) K-means clustering of 127 genes with an FDR q < 0.05 comparing the Uhrf1 fl/fl and Uhrf1 +/+ iTreg populations on day 12 in which UHRF1 was deleted after FOXP3 induction (delayed) with k = 2. (F) MA plot comparing gene expression of delayed Uhrf1 +/+ and Uhrf1 fl/fl iTregs on day 12 of culture. Genes of interest are annotated. (G) Enrichment plot of the GSE14415_INDUCED_TREG_VS_TCONV_UP, GSE14415_INDUCED_TREG_VS_TCONV_DN, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_UP, GSE37605_TREG_VS_TCONV_C57BL6_FOXP3_FUSION_GFP_UP, GSE13306_TREG_VS_TCONV_UP gene sets ( p < 0.05, FDR q < 0.25) generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture. (H) PCA of 81,179 differentially methylated cytosines identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (I) Cumulative distribution function plot of differentially methylated cytosines expressed as β scores, with 0 representing unmethylated and 1 representing fully methylated; a shift in the cumulative distribution function up and to the left represents relative hypomethylation. * q < 0.05 according to two-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Cell Culture, Standard Deviation, Gene Expression, Generated, Control, Methylation

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) MA plot comparing gene expression of Uhrf1 +/+ iTregs (control) with CD4 + Foxp3 -GFP + nTregs on day 5 of culture. Genes of interest are annotated. (B) Enrichment plots of the HALLMARK_MYC_TARGETS_V1, HALLMARK_E2F_TARGETS, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_MTORC1_SIGNALING, HALLMARK_IL2_STAT5_SIGNALING, and HALLMARK_KRAS_SIGNALING_UP gene sets generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Gene Expression, Control, Generated

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A-F) Total numbers of CD45 + (A), CD3 + (B), CD4 + CD8 − (C), CD4 − CD8 + (D), CD11b − CD64 + CD11c + SiglecF + (E), and CD11b + Ly6G + (F) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 16), or U hrf1 fl/fl iTregs. (n = 16) (G) Total number of CD326 + CD31 − CD45 − (epithelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 7), Uhrf1 +/+ iTregs (n = 15), or U hrf1 fl/fl iTregs (n = 16). (H) Total number of KRT5 + CD326 + epithelial cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 7), Tconv (n = 7), PBS (n = 7), Uhrf1 +/+ iTregs (n = 13), or U hrf1 fl/fl iTregs (n = 14). (I) Total number of CD326 − CD31 + (endothelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 14), or U hrf1 fl/fl iTregs (n = 15). Data presented as mean and SD. ns = not significant by multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% test.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: MANN-WHITNEY