Science - USA (2022-06-03)

Suoet al., Science 376 , eabo0510 (2022) 3 June 2022 10 of 15

TRAV genesTRAJ genes

clone of

proliferating

DP

transition stage

transition stage

Unconventional

T cell

Conventional

T cell

cortex medulla

nearby

cTEC

positive selection

T–T interaction

positive selection

T–TEC interaction

recombination

B

D E

F G

A

Proportion of scVDJseq cells (%)

Conventional T cells Unconventional T cells

cell density

C

TRAV TRAJ

5’ 3’

V J

** ** ** ******** * ** **

Unconventional

T cells

Conventional

T cells

*

*

Unconventional T cells

Conventional T cells

Gene segment preferentially used in

* *

*** *

TRGV TRGJ TRDV TRDD TRDJ

development

genomic position

iPSC

ATO

T cells

Hematopoietic

induction

(2 weeks)

T cell

differentiation

(7 weeks)

Mesoderm

induction

(3.5 days)

Mouse stromal

cells with

human DLL4

D0 wk5 wk7wk3

PLZF

Fig. 6. Deep characterization of human unconventional T cells.(A) Proportions
of cells expressing pairedgdTCR, pairedabTCR, both, or neither. The
proportions were calculated over cells that had both single-cellabTCR and
gdTCR sequencing. Expression of neither pairedabTCR nor pairedgdTCR in
some cells could be due to dropouts in single-cell TCR sequencing because

50% of these contained orphan VDJ or VJ chains ofabTCR orgdTCR
(fig. S28A). (B) Heatmap showing the percentage of eachgdTCR gene segment
present in different T cell subtypes. Differential usage between cell subtypes
was computed using the chi-squared test. Gene segments with Benjamini-
Hochberg–adjustedP values < 0.05 are marked with asterisks. (C) Heatmap

showing the proportion of each TCRagene segment present in different T cell

subtypes. The gene segment usage in unconventional T cells and conventional

T cells was compared using logistic regression, controlling for donors and

organs. Gene segments with Benjamini-Hochberg–adjustedP values < 0.05

are marked with magenta asterisks for preferential usage in unconventional

T cells and green asterisks for preferential usage in conventional T cells.

(D) Principal component analysis plot summarizing TRAV, TRAJ, TRBV, and

TRBJ gene segment usage proportion in different T cell subtypes. Each dot

represents a sample of at least 20 cells, with dot size representing the cell count.

The centroid of each cell type is shown as a filled circle, and 80% confidence

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