Nature - USA (2020-01-23)

Article

% IGHD of B−cells

Non−responderResponder

IGHD P = 0.19

% IGHG of B−cells

IGHG P = 0.56

0

1

2

3

4

Iliac lymph node Axillary lymph nodeAxillary lymph nodeAxillary lymph node External iliac lymph node Acral primary SpleenTerminal Ileum Cutanoues met Axillary lymph nodeAxillary lymph nodeSubcutaneous leg lesionSmall bowel Adrenal gland metastasisBulky (L) axillary metastasis SkinNecrotic L axillary lymph nodesLymph nodeLymph nodeR adrenal metastasisTransanal rectal massSoft tissueSkinSkinSkinSkinLymph node

Mel94Mel79Mel80Mel89Mel72Mel84Mel60Mel74Mel88Mel82Mel81Mel58Mel78Mel102Mel112Mel105Mel106Mel103Mel116Mel110Mel478Mel126

Mel129pbMel129paMel121

Mel04Mel128

Activated B cellImmature B cell

Memory B cellPlasmacellUP_BcellDN

a.

012 3 456

Plasma cells

IGLL1/5-/CD69-

IGLL1/5-/CD69+

IGLL1/5+/CD69-

IGLL1/5+/CD69+

Absolute expr.

% CD69+ B cells

Non−responderResponder

CD69, P = 0.0037

0

20

40

60

80

100

% IGLL5+ B cells

IGLL5, P = 0.17

0

10

20

30

40

50

Non−responderResponder

IGLL5-/CD69+,

P = 0.0098

Non−responderResponder

% IGLL5-/CD69+ B cells

0

20

40

60

80

100

anti-CTLA4

anti-CTLA4+PD1

anti-PD1

BACH2

MEF2BMYC

MEF2CPOU2AF1

IRF4

CCND2CD19

AIM2FCER2

TCL1AREL

EZH2ICOSLG

CD40MS4A1

XBP1JAM3

NOTCH2CD83

PDCD1CD86

CXCR4LAMP3

SELLCXCR5

CCR7CXCL13

CCL21CCL19

HLA−BHLA−A

CD74Mean_HLAD

HLA−DRAMKI67

IGLL1IGLL5

JUNBNR4A2

FOSCD69

DZLZ

AICDABCL6

PRDM1SDC1

PlasmacellUP_BcellDNMemory B cell

Immature B cellActivated B cell

Tumor

Mel04.3

Mel102Mel103

Mel105

Mel106Mel110

Mel112

Mel116Mel121.1

Mel126

Mel128Mel129pa

Mel129pb

Mel478Mel58

Mel60

Mel72Mel74

Mel78

Mel79Mel80

Mel81Mel82

Mel84Mel88

Mel89

Mel94^0

20

40

60

80

100

0

20

40

60

80

100

Non−responderResponder

CCND3

HLA−C

TLS hallmark

genes

GC initiation

genes

GC genes

DZ proliferation

B cell related

genes

Tumors

b.

c.

CD79B

CD1D

CCR6

LAT

SKAP1

CETP

EIF1AY

RBP5

PTGDS

BACH2 % IGHG^2

GCgenes initiation (^4)
CCL19
CCL21
CXCL13
CCR7
CXCR5
SELL (CD62L)
LAMP3
0246810
02468
CD69
CD83
0246810
0246810
CD69
CXCR4
CD69-
CD69+
CD83+
CD83-
CD83+
CD83-
P = 1.4x10-8
correlation = 0.31
CD69-
CD69+
CXCR4+
CXCR4-
CXCR4-
CXCR4+
P = 2.6x10-6
correlation = 0.37
d.
e. f.
Extended Data Fig. 3 | scRNA-seq analysis of tumour-associated B cells.
a, Box plots of gene-expression scores, based on different B cell developmental
states in 812 B cells from 27 tumours from a previous study^18. b, Heat map of
selected genes across all 812 B cells. IGLL5and CD69 were two of the five genes
with highest expression variation across all B cells. The heat map is sorted on
IGLL5 and CD69 expression, excluding the cells that displayed increased
expression of the plasma-cell signature. Genes showing a Pearson correlation

0.4 to IGLL5 or CD69 expression are also indicated. SDC1 and PRDM1 mark
plasma cells, BCL6 and AICDA mark germinal centres, HLA-DRA mark MHC-II
and HLA-A, HLA-B and HLA-C mark MHC-I. TL S-hallmark genes, germinal-
centre-related genes and other B cell genes are also indicated. c, Extracting the
single B cell RNA-seq data from a previous study^17 using pretreatment samples
(n = 16). The fraction of CD69+ B cells was higher in responders to ICB than in
nonresponders (n = 8), but the fraction of IGLL5+ B cells was not. The fraction of
IGLL5− CD69+ cells was also higher in responders. Plots of fraction of IGHD+ and
IGHG+ B cells in relation to response to ICB therapy. P values from two-sided
Wilcoxon test. In a, the centre lines in the box plot represent the median, the
box limits represent the lower and upper quartiles, and the whiskers extend to
the most extreme values within 1.5× IQR. d, Pearson correlation between
expression of CD69 and germinal centre genes (CD83 and CXCR4) in data from a
previous study^18. Pie charts display the fact that the fraction of CD83+ and
CXCR4+ B cells is increased among CD69+ B cells. Expression > 1 was used as a
cut-off for being present. Seven hundred and fifty-three B cells without a
present plasma-cell signature were analysed. P value from two-sided Fisher’s
exact test. e, f, Heat map of gene-expression values corresponding to our TLS
signature (e) and TLS-hallmark genes from the literature (f). Blue corresponds
to increased expression. Mal., malignant cells. In e, f, single cells from the seven
cell types on the left are from ref.^18 , and from the four cell types on the right are
from ref.^17.