Science - USA (2021-07-16)

Jacobsenet al.,Science 373 , eabe5146 (2021) 16 July 2021 8 of 13

F Foxp3+ vs. Foxp3– cells in mixed clones

A Photoactivated T cells + naïve adoptive transfer

Cluster 1 “Tfh1”

Cluster 2 “Tfh2”

Cluster 3 “Treg/resting”

UMAP 2

UMAP 1

−2.5

0.0

2.5

5.0

7.5

−3 0 3

Cluster 4 “Activated Treg”

Cluster 5 “CD8+”

Cluster 6 “Cycling”

B

UMAP 2

UMAP 2

RFP+

Other

−2.5

0.0

2.5

5.0

7.5

−3 0 3

GC photoactivated

RFP+

Other

−2.5

0.0

2.5

5.0

7.5

T-zone photoactivated

−3 0 3

Naïve transfer

Other

−2.5

0.0

2.5

5.0

7.5

UMAP 1

−3 0 3

Naïve transfer (GFP+)

T-Zone

(72)

GC

(276)

NT

(51)

0.00

0.25

0.50

0.75

1.00

% of Foxp3

+ in cluster

Naïve_xfer_UP

P = 0.40 (–0.54)

Naïve_xfer_DN

P = 0.026 (0.91)

C RFP+ Cluster 1+2 vs.

RFP+ Cluster 4

Wing_GC-Tfr_vs_Tfr_UP

P = 4.1e–19 (1.29)

−0.10

0.00

0.10

Score

Wing_GC-Tfr_vs_Tfr_DN

P = 1.5e–10 (–0.67)

−0.1

0.0

0.1

0.2

Cluster 1+2

Cluster 4

Score

D “Naïve transfer” Foxp3-GFP+ Tfh vs. Foxp3-RFP– Tfh

Wing_

GC-Tfr_vs_Tfh_UP

P = 3.4e−8 (–0.97)

Wing_

GC-Tfr_vs_Tfh_DN

P = 5.5e–19 (1.9)

GSE20366_Treg_vs_

naïve_CD4_Tcell_UP

P = 1.7e–11 (–1.4)

GSE20366_Treg_vs_

naïve_CD4_Tcell_DN

P = 2.9e–13 (1.4)

0.0

0.1

−0.2

0.0

0.2

Score

Expression

Foxp3 Naïve transfer

• Naïve Tfh
  Foxp3 transfer


• 
  

  Naïve Tfh
  Foxp3 transfer

  
  


• 
  

  Naïve Tfh
  Foxp3 transfer

  
  


• 
  

  Tfh

  
  

−0.1

0.0

0.1

−0.1

0.0

0.1

Cd40lg

P = 0.0043

Foxp3 Naïve transfer

• Tfh

0

1

2

3

Il21

P = 0.0012

Foxp3 Naïve transfer

• Tfh

0

1

2

3

E Clones containing both Foxp3-RFP+ and Foxp3-RFP– cells

−2.5

0.0

2.5

5.0

7.5

−3 0 3

UMAP 2

UMAP 1

RFP+

RFP–

Background

Same clone

Wing_GC-Tfr_vs_Tfh_UP

P = 0.049 (–0.85)

Wing_GC-Tfr_vs_Tfh_DN

P = 0.0063 (1.14)

Expression

Score

Cd40lg

P = 0.21

Expression

Score

RFP

• RFP

+

RFP

• RFP

+

RFP

• RFP

+

−0.25

0.00

0.25

0.50

−0.1

0.0

0.1

0.2

−0.02

0.00

0.02

0

1

2

3

−0.25

0.00

0.25

0.50

0.75

Il21

P = 0.021

0

1

2

3

Fig. 6. scRNA-seq of GC-resident T cells.Total T cells (Foxp3RFP+and
Foxp3RFP–combined) from single photoactivated GCs were sorted either as
B220–/CD4+/PA+or as B220–/TCRb+/PA+(which also includes CD8+T cells) in
different samples at day 10 or 20 after primary immunization with NP-OVA. For
comparison, we also sorted Tregsphotoactivated in the T-zone of an unimmu-
nized mouse, sorted as B220–CD4+GFP+RFP+and Foxp3+CXCR5+PD-1hiTFHcells
derived from transferred naïve precursors as in Fig. 5. (A) Uniform manifold
approximation and projection (UMAP) plot showing clustering of T cells
according to whole-transcriptome analysis. (B) Distribution of different subsets
of Foxp3-reporter+cells in UMAP space. Cells in color are those that are
Foxp3-reporter+in the sample indicated in the graph title. All other analyzed cells
are shown in gray. The bar graph shows the distribution of T-zone and
GC RFP+cells among clusters 1 to 3. NT, naïve transfer. (C) Expression of

selected gene signatures by Foxp3RFP+and Foxp3RFP–cells within TFHclusters

1 and 2. (D) Expression of selected genes or gene signatures by Foxp3+TFHcells

derived from transferred naïve precursors compared with photoactivated

Foxp3–TFHcells. Only cells from day 20 postimmunization are included in the

Foxp3–TFHgroup.“GSE20366”signatures are from the Broad Institute’s

MSigDB database. (E) Distribution of clonal expansions (defined as TCRs

detected more than once in the same GC) containing both RFP+and RFP–cells

(“mixed clones”). Cells carrying identical TCRs are linked by lines. (F) Expression

of selected genes or gene signatures by Foxp3-RFP+and Foxp3-RFP–cells

within mixed clones. In (A) to (F), each symbol represents one cell.Pvalues

are for Wilcoxon signed-rank test. Numbers in parentheses are Cohen’sdfor

effect size. The number of GCs included and the number of independent

experiments are indicated in table S1.

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