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).