Nature - USA (2020-01-02)

Nature | Vol 577 | 2 January 2020 | 97

h

–log

( 10

P value)

log 2 -transformed fold change

0

100

200

300

400

−2 −1 0 1 2

TNFSF8

CD8A NKG7

CD8B

CD6 IL21R

IL2RA CD40LGCD4

RORC

g

IV

IDlow

IDhigh

AE

Naive

–0.250.000.250.50

Week 13 T cells Week 25 T cells

–0.2 00 0.00 .200.40

Stimulated Unstimulated

Module score

GNLY

PIK3AP1

PRF1

APOL2

GPR68 TNFRSF9TNFRSF4

IFNG

HERC6

SLC29A1

STK17B PRDM1 TNFRSF8

TPT1

f

Percentage

above

Percentage

above

36.4%

9.46%

5.18%

6.46%

0.00%

24.6%

5.43%

3.92%

7.46%

0.00%

Module 2 (0.01)

Module 5 (0.03)

Module 7 (0.08)

Module 3 (0.80)

Module 6 (0.18)

Module 1 (<1 × 10 –5)

Module 4 (P = 0.13)

Week 13 stimulated T cells

Module 2 (3.0 × 10 –4)

Module 4 (0.36)

Module 6 (0.08)

Module 7 (0.63)

Module 5 (9.2 × 10 –4)

Module 3 (0.12)

Module 1 (0.0047)

Week 25 stimulated T cells

–1.5 1.5

Row Z-score

log

(cells per BAL collection) 10

Weeks after BCG immunization

a Total cell

4

8

6

T cell CD4 CD8 Vγ^9 +^ γδ Vγ^9 –^ γδ MAIT

iNKT

2

6

4

P 8 16 24

Macrophage

4

8

6

P 8 16 24

NK

P 8 16 24

B

P 8 16 24

Neutrophil

P 8 16 24

mDC

P 8 16 24

pDC

P 8 16 24

IDlow IDhigh IV AE AE/ID IDlow IDhigh IV AE AE/ID

IFN

γ, IL-2, TNF orIL-17 (%)

IFN

γ, IL-2, TN

F or IL-17

(%)

(log

(cell number)) 104

6

2

P 4 24 P 4 24 P 4 24 P 4 24 P 4 24 P 4 24 P 4 24 P 4 24 P 4 24 P 4 24

P 8 16 P 8 16 P 8 16 P 8 16 P 8 16 P 8 16 P 8 16 P 8 16 P 8 16 P 8 16

40

80

20

60

0

2

6

4

1

3

0

1.0

0.5

1.5

0.0

20

0

10

4

6

2

Weeks after BCG immunization

BCG regimen

d

c

e

b

IDhigh

IV

AE

IDlow

AE/ID

<0.0010.022

<0.001<0.001

0.030 0.004

0.015 0.031 0.014 0.017 <0.001 <0.001

<0.0010.018

<0.001 <0.001 <0.001

0.006<0.001

0.0030.003 <0.001

Naive IDlow IDhigh IV AE

Fig. 1 | Cellular composition and immune analysis in blood and BAL after
BCG vaccination. a, Number of cells (geometric mean) per BAL collection for
leukocyte populations in each vaccine group before (pre, P) and up to 24 weeks
after BCG; Supplementary Data 2 shows individual NHPs and statistical
comparisons. Data are from cohorts 1–4 (n = 11–13 macaques per group as
outlined in Extended Data Fig. 1) except at weeks 2, 20 and 24 (cohort 4 only,
n = 3). Vγ 9+/−, Vγ9+/− γδ T cells; MAIT, mucosal-associated invariant T cells; mDC,
myeloid dendritic cells; NK, natural killer cells; iNKT, invariant natural killer
cells; pDC, plasmacytoid dendritic cells. b, c, Percentage of memory CD4 (b) or
CD8 (c) T cells in PBMCs producing IFNγ, IL-2, TNF or IL-17 after PPD stimulation
in vitro. Shown are individual and median (horizontal bar) responses for NHPs
in challenge study (cohorts 1–3, n = 8–10 macaques) at weeks 4 (peak) and 24
(time of challenge) after BCG vaccination. d, e, Percentage (top) and number
(bottom) of cytokine+ memory CD4 (d) and CD8 (e) T cells in the BAL before and
up to 16 weeks after BCG vaccination. Shown are individual (grey lines) and

mean (coloured lines) responses for challenge cohorts (n = 8–10 macaques).

Each group was compared to IDlow at weeks 4 and 24 for PBMCs (one-way

A N OVA ; P values are Dunnett’s multiple comparison test) or weeks 8 and 16 for

BAL (Kruskal–Wallis test; P values are Dunn’s multiple comparison test).

f–h, Single-cell transcriptional analysis of BAL cells at weeks 13 and 25 after BCG

vaccination (cohort 4; n = 3 per group). f, Z-scored heat maps of the average

cellular score for modules identified in week 13 PPD-stimulated T cells at weeks

13 and 25 after BCG vaccination. Red P values indicate modules uniquely

elevated in the IV BCG group (one-way ANOVA). g, Distributions of module 2

expression in unstimulated and stimulated T cells at weeks 13 and 25 for each

group. Percentage module 2-positive is shown; positivity (dashed line) defined

as 2 s.d. above the mean score of the unvaccinated (Naive) NHPs. h, Volcano

plot showing differentially expressed genes between T cells positive and

negative for module 2 at week 13 (P values calculated using the likelihood ratio

test with Bonferroni correction).