Figure 1. Loss of NCOR1 leads to a relative increase in CD44^hiCD62L^– effector Treg cells.

(a) Flow cytometric analysis of splenocytes isolated from wild-type (WT) and NCOR1-cKO mice showing CD44 and CD62L expression in CD4⁺FOXP3⁺ cells at steady-state. (b) Diagrams showing the percentage of CD44^hiCD62L^– (left) and CD44^loCD62L⁺ (right) CD4⁺FOXP3⁺ cells of all mice analyzed as described in (a). (c) Total cell numbers of CD44^hiCD62L^– (left) and CD44^loCD62L⁺ (right) CD4⁺FOXP3⁺ cells of all mice analyzed are described in (a). (d) Experimental immunization strategy: mice were injected s.c. with nitrophenol keyhole limpet hemocyanin (NP-KLH) and draining lymph nodes (LNs) analyzed six days later. (e) Flow cytometric analysis of cells isolated from draining LN of NP-KLH-immunized WT and NCOR1-cKO mice showing the expression of PD-1, CXCR5, and FOXP3 on CD4⁺CD44^hi cells. (f) Diagrams showing the percentage of T follicular helper (Tfh) cells (CD4⁺CD44^hiPD1⁺) (left) and T follicular regulatory (Tfr) cells (CD4⁺CD44^hiPD1⁺CXCR5⁺FOXP3⁺) (right) of all mice analyzed as described in (d,e). (g) Total cell numbers of Tfh cells (CD4⁺CD44^hiPD1⁺CXCR5⁺) (left) and Tfr cells (CD4⁺CD44^hiPD1⁺CXCR5⁺FOXP3⁺) (right) of all mice analyzed as described in (d,e). (h) Flow cytometric analysis of cells isolated from draining LN of NP-KLH-immunized WT and NCOR1-cKO mice showing the expression CD25 in CD4⁺CD44^hiPD1⁺CXCR5⁺FOXP3⁺ Tfr cells. (i) Percentage of CD25 expressing CD4⁺CD44^hiPD1⁺CXCR5⁺FOXP3⁺ Tfr cells of all mice analyzed as described in (h). (a,e,h) Numbers indicate the percentages of cells in the respective quadrants or gates. (a–c) Cells were pre-gated on CD4 and FOXP3. (b,c,f,g,i) Each symbol indicates one mouse. Horizontal bars indicate the mean. *p<0.05, **p<0.01, and ***p<0.001; Unpaired two-tailed Student’s t-test. Data are representative (a,e,h) or show the summary (b,c,f,g,i) of at least eleven (b,c), twelve (f,g) or five (i) mice that were analyzed in at least two independent experiments.

Figure 1—figure supplement 1. Effector markers are upregulated in NCOR1-deficient Treg cells.

(a) Diagrams showing a summary of the percentage of CD44^hiCD62L^– (left) and CD44^loCD62L⁺ (right) CD4⁺FOXP3⁺ cells from lymph nodes (LNs) of wild-type (WT) and NCOR1-cKO mice. (b) Diagrams showing a summary of the percentage of CD44^hiCD62L^– (left) and CD44^loCD62L⁺ (right) CD4⁺FOXP3⁺ cells from mesenteric LNs (mLNs) of WT and NCOR1-cKO mice. (c) Histograms displaying expression of KLRG1, CD69, CD25, ICOS, GITR, and CTLA4 in WT and NCOR1-cKO Treg cells from spleen, LNs and mLNs. The dotted lines indicate expression on conventional CD4⁺ T cells (Tconv). (d) Diagrams showing the summary of all experiments performed as described in (c). For the calculation of GITR and CTLA4 relative gMFI levels, the average expression levels in WT cells (n=4 in experiment 1 and n=3 in experiment 2) were set as 1 and relative gMFI on individual NCOR1-cKO Treg samples (n=4, and n=3) was calculated. (a,b,d) Each symbol indicates one mouse. Horizontal bars indicate the mean. *p<0.05, **p<0.01, and ***p<0.001; unpaired two-tailed Student’s t-test. Data are representative (c) or show a summary (a,b,d) of at least seven mice analyzed in at least two independent experiments.

Figure 1—figure supplement 2. Characterization of FOXP3⁺ Treg cells isolated from NCOR1-cKO mice.

(a) Flow cytometric analysis of wild-type (WT) and NCOR1-cKO splenocytes showing expression of Ki67 in CD4⁺ FOXP3⁺ cells at steady state. (b) Summary showing percentage (left) and expression (gMFI) (right) of Ki67 in CD4⁺FOXP3⁺ cells of all mice analyzed as described in (a). (c) Summary showing the percentage of viable FOXP3⁺ splenocytes cultured in the presence of anti-CD3/anti-CD28 over the course of 3 days. (d) Diagrams showing the percentages of CD44^hiCD62L^– (left) and CD44^loCD62L⁺ (right) conventional CD4⁺ T cells (FOXP3^–) (Tconv) isolated from the spleens of WT and NCOR1-cKO mice. (e) Flow cytometric analysis showing FOXP3 and CD25 expression in WT and NCOR1-cKO CD4SP thymocytes. (f) Diagram showing the percentage of FOXP3^–CD25⁺, FOXP3⁺CD25^– and FOXP3⁺CD25⁺ CD4SP thymocytes of all mice analyzed as described in (e). (g) Diagrams showing the percentages of thymic CD25^–CD44^hiCD62L^– and CD25⁺CD44^hiCD62L^– subsets on FOXP3⁺ CD4SP cells from WT and NCOR1-cKO mice. (h) Summary showing the percentage of phospho-ERK^T202/Y204, phospho-AKT^S473 and phospho-S6^S240/244 expression in FOXP3⁺ splenocytes cultured in the presence of anti-CD3/anti-CD28 over the course of 24hr. (a) Numbers indicate the percentages of cells positive for Ki67. (e) Numbers indicate the percentages of cells in the respective quadrants. (b,d,f,g) Each symbol indicates one mouse. Horizontal bars indicate the mean. *p<0.05, **p<0.01, and ***p<0.001. (b,d,f,g) Unpaired two-tailed Student’s t-test. (c,h) Two-way ANOVA analysis followed by Tukey’s multiple-comparisons test. Data are representative (a,e) or show a summary (c,d,f,g,h) of at least two independent samples (c,h) or at least seven (f,g) or eleven (d) mice that were analyzed in one (c,h) or at least two (d) or three (f, g) independent experiments.

Figure 1—figure supplement 3. Gating strategies for flow cytometric analysis.

(a) Gating strategy for identification of effector (CD44^hiCD62L^–) and naïve (CD44^loCD62L⁺) Treg cells: After exclusion of debris (FSC-A/ SSC-A), doublets (FSC-H/ FSC-W) and dead cells (using Viability Dye eFluor 506 or 780), cells were distinguished via their expression of CD19 (BV605) and TCRβ (BV711), CD4 (PE-Cy7) and CD8 (AF700) and FOXP3 (APC). Cells were further gated based on their expression of CD44 (PerCP-Cy5.5) and CD62L (APC-Cy7). (b) Gating strategies for identification of Tfh (CXCR5⁺PD1⁺CD44^hi) and Tfr (CXCR5⁺PD1⁺CD44^hiFOXP3⁺) cells: Debris, doublets, and dead cells were excluded as described in (a). Cells were distinguished via their expression of CD19 (BV605) and TCRβ (BV711), CD4 (PE-Cy7) and CD8 (AF700), CD44 (PerCP-Cy5.5), CXCR5 (BV421) and PD-1 (BV786) and FOXP3 (APC). (c) Gating strategy for the adoptive transfer colitis experiment: After exclusion of debris (FSC-A/ SSC-A) and doublets (FSC-H/ FSC-W) cells were distinguished via their expression of CD4 (PE-Cy7) and their viability using the Viability Dye eFluor 506. Cells were further gated based on their expression of CD45.1 (PE/ Dazzle) and FOXP3 (APC). (d) Gating strategy for the CRISPR-Cas9 mediated knockout experiment: After exclusion of debris (FSC-A/ SSC-A) and doublets (FSC-H/FSC-W) cells were distinguished via their expression of FOXP3 (eFluor 450) and MYC (AF647) or via their expression of the markers CD45RA (PE), CD45RO (APC-Cy7), and CD27 (AmCyan).