Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

Eddie Cano-Gamez, Blagoje Soskic, Theodoros I. Roumeliotis, Ernest So, Deborah J. Smyth, Marta Baldrighi, David Willé, Nikolina Nakic, Jorge Esparza-Gordillo, Christopher G. C. Larminie, Paola G. Bronson, David F. Tough, Wendy C. Rowan, Jyoti S. Choudhary, Gosia Trynka

Study ID


Total cells


Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

Author's cell type

TN (Th17)
4675 cells
TN (iTreg)
4383 cells
TCM2 (Th17/iTreg)
3860 cells
TN (Th2)
3582 cells
TEM (Th17/iTreg)
2998 cells
TN (Th17/iTreg)
2618 cells
TCM2 (Th0)
2596 cells
TN (resting)
2421 cells
TN (Th0)
2410 cells
TCM (resting)
1915 cells
TCM1 (Th0)
1909 cells
1650 cells
1318 cells
TEM (Th0)
1297 cells
1174 cells
TCM1 (Th17/iTreg)
1041 cells
TEMRA (Th17/iTreg)
981 cells
nTreg (Th0)
959 cells
TEM (resting)
456 cells
392 cells
nTreg (resting)
253 cells
TEMRA (resting)
224 cells

Average age

56.4 years
43112 cells

Cell class

Memory CD4 T cell (CD25− CD45RA + CD45RO−)
22167 cells
Naive CD4 T cell (CD25− CD45RA + CD45RO−)
20945 cells

Cell cycle phases

22590 cells
11914 cells
8608 cells

Cell type

naive CD4 T cell
20089 cells
central memory CD4 T cell
11321 cells
effector memory CD4 T cell
4751 cells
CD4 T cell
4142 cells
TEMRA CD4 T cell
1597 cells
1212 cells


43112 cells

Cytokine stimulation

TGF-β1 (5 ng/ml), IL-2 (10 ng/ml)
12719 cells
12578 cells
IL-6 (50 ng/ml), IL-23 (20 ng/ml), IL-1β (10 ng/ml), TGF-β1 (5 ng/ml), Anti-human IL-4 antibody (1 ug/ml), Anti-human IFN-γ antibody (1 ug/ml)
10882 cells
IL-4 (10 ng/ml), Anti-human IFN-γ antibody (1 ug/ml)
6933 cells

Developmental stage

43112 cells


43112 cells

Louvain clustering

Cluster 7
6678 cells
Cluster 8
5810 cells
Cluster 5
5615 cells
Cluster 1
5032 cells
Cluster 9
4807 cells
Cluster 11
3940 cells
Cluster 10
3781 cells
Cluster 6
3062 cells
Cluster 4
2311 cells
Cluster 3
1838 cells
Cluster 2
238 cells


43112 cells

Sample ID

Donor 3
11553 cells
Donor 1
11320 cells
Donor 4
11009 cells
Donor 2
9230 cells

Sequencing platform

Chromium v2
43112 cells

Storage technique

43112 cells

Subject ID

Donor 3
11553 cells
Donor 1
11320 cells
Donor 4
11009 cells
Donor 2
9230 cells

T cell stimulation state

12719 cells
10882 cells
7309 cells
6933 cells
Unstimulated cells
5269 cells

Tcr stimulation

Anti-CD3/anti-CD28 beads 1:2 to T cells
37843 cells
5269 cells


cultured blood
43112 cells