stm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1

Supplementary Materials for

Identification of tetranectin-targeting monoclonal antibodies to treat potentially

lethal sepsis

Weiqiang Chen, Xiaoling Qiang, Yongjun Wang, Shu Zhu, Jianhua Li, Ariella Babaev, Huan Yang, Jonathan Gong, Lance Becker, Ping Wang, Kevin J. Tracey, Haichao Wang*

*Corresponding author. Email: hwang@northwell.edu

Published 15 April 2020, Sci. Transl. Med. 12, eaaz3833 (2020)

DOI: 10.1126/scitranslmed.aaz3833

The PDF file includes:

Materials and Methods Fig. S1. Immunoblotting and genotyping analysis of TN KO mice. Fig. S2. Survey of TN protein abundance in various tissues. Fig. S3. Parallel reduction of lung and serum TN content during lethal endotoxemia. Fig. S4. Expression and purification of recombinant TN. Fig. S5. Recombinant murine TN conferred a dose-dependent protection against lethal sepsis. Fig. S6. Epitope mapping and specificity of representative mAbs raised against recombinant human TN. Fig. S7. Cross-reactivity and epitope mapping of a panel of P5-reacting mAbs. Fig. S8. Epitope sequence homology between different mammalian species. Fig. S9. Characteristics of a panel of human TN-specific mAbs. Fig. S10. IgG isotype controls did not affect sepsis lethality. Fig. S11. Distinct effects of P2- and P5-reacting mAbs on sepsis-induced TN depletion. Fig. S12. Divergent effects of mAb8 and mAb9 on sepsis-induced systemic KC accumulation. Fig. S13. TN enhanced HMGB1 uptake and possible degradation by macrophage cultures. Fig. S14. mAb8 and dynasore inhibited the TN/HMGB1-induced increase of trypan blue dye uptake in macrophage cultures. Fig. S15. Proposed model for the mAb8-mediated protection against lethal sepsis. Table S1. Reagent sources. Table S2. Demographics of 44 normal healthy controls and 45 septic patients.

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/12/539/eaaz3833/DC1)

Data file S1 (Microsoft Excel format). Primary data.

Materials and Methods

Genotyping

To verify the genotypes of wild-type (WT) and TN KO mice, tail biopsies was digested in

Direct-PCR lysis Reagent (Cat. No 102-T, Viagen Biotech, Inc.) containing 0.4 µg/ml proteinase

K (Cat. No EO0491, ThermoFisher Scientific), and lysate containing genomic DNA was

amplified by PCR reaction using the following primers: forward WT primer CAA AAA CCA

CAC ACT CCA TCT G; reverse WT primer CTT AGT ATC TAC CAC TCC TGT CTG AGG;

forward KO mutant primer CGG TCG CTA CCA TTA CCA GT; reverse KO mutant primer

TGT GTT GTA GTC CAG CAG AGG, under the following conditions: 95ºC 3 min; followed

by 37 cycles of 95ºC for 15 sec and 60ºC for 15 sec, 72ºC, 15 sec. The PCR products were

resolved on a 2% agarose gel and visualized by ethidium bromide staining.

Preparation of recombinant HMGB1 and TN proteins

The cDNA encoding for rat HMGB1 was cloned into a pCAL-n vector, and the recombinant

CBP-HMGB1 (rHMGB1) was expressed in E. coli BL21 (DE3) pLysS cells as previously

described (4). Recombinant HMGB1 containing a ~3 kDa calmodulin-binding peptide tag (CBP-

HMGB1 fusion protein, 33 kDa) was expressed in E. coli, and purified to remove contaminating

endotoxin by Triton X-114 extraction as previously described (50). Recombinant human TN

corresponding to amino acids 22-202 (without the 21-amio acid leader signal sequence) with a

C-terminal histidine tag was expressed in E. coli BL21 (DE3) pLysS cells, and purified by

histidine-affinity and Triton X-114 extraction to remove contaminating endotoxins. Recombinant

TN and HMGB1 proteins were tested for LPS content by the chromogenic Limulus amebocyte

lysate assay (Endochrome; Charles River), and the endotoxin content was less than 0.01 U per

microgram of recombinant proteins.

Cellular HMGB1 and TN uptake

Highly purified recombinant HMGB1 and TN proteins were labeled with either Alexa Fluor 555

or Alexa Fluor 488 using the appropriate labeling kits (Cat. # A30007 or Cat. # A30006, Thermo

Fisher Scientific) according to the manufacturer’s instructions. Murine macrophage-like RAW

264.7 cells on cover slips were incubated with unlabeled or Alexa Fluor 555-labeled HMGB1 in

the absence or presence of unlabeled or Alexa Fluor 488-labeled TN at 37⁰C for 2 h. An

endocytosis inhibitor, Dynasore (8.0 µM), or mAb8 (65 µg/ml) were added 30 min before the

addition of HMGB1 or TN. After extensive washing with 1 x PBS, cells were fixed with 4%

formalin for 20 min at room temperature and mounted on slides using Vectashield Mounting

Medium for Fluorescence with DAPI (Vector Laboratories, Inc., Cat. # H-1200) for nuclei

staining (blue). Endocytic uptake of Alexa Fluor 555-labeled HMGB1 (red) or Alexa Fluor 488-

labeled TN (green) was visualized under the Olympus IX51 Inverted Fluorescence & Phase

Contrast Tissue Culture Microscope. To quantitate cellular uptake of HMGB1 and TN,

macrophage cultures were incubated with recombinant HMGB1 and TN either individually or in

combination, at 37⁰C for 2 h. After extensive washing, cellular concentrations of HMGB1 and

TN were measured by Western blotting analysis with reference to a housekeeping protein, β-

actin.

Western blotting

The concentration of TN in human or murine serum was determined by Western blotting analysis

using commercial rabbit mAb against the C-terminus of human TN (Abcam) or our homemade

murine mAb (mAb8) against recombinant human TN. The cellular and extracellular

concentrations of TN and HMGB1 in murine macrophage and human monocyte cultures were

determined by Western blotting analysis using rabbit polyclonal or monoclonal antibodies. Equal

amounts of total cellular protein or equivalent volumes of cell-conditioned culture medium were

resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene

difluoride (PVDF) membranes. After blocking with 5% nonfat milk, the membranes were

incubated with the appropriate antibodies (anti-TN, 1:1000; anti-ASC, 1:1000; anti-β-actin,

1:5000; anti-HMGB1, 1:1000) overnight. Subsequently, the membranes were incubated with the

appropriate secondary antibodies, and the immune-reactive bands were visualized by

chemiluminescence. The relative band intensity was quantified using the UN-SCAN-IT Gel

Analysis Software Version 7.1 (Silk Scientific Inc.).

Fig. S1. Immunoblotting and genotyping analysis of TN KO mice. (A) Western blotting

analysis of serum and lung TN from wild-type (TN+/+

), heterozygous (TN+/

), and homozygous

(TN-/-

) knockout mice. SDS-PAGE gel analysis confirmed equivalent sample loading. (B)

Genotyping of wild-type and TN knockout mice by PCR. Genomic DNA samples were isolated

from mouse tail biopsies and amplified by PCR. The PCR products of wild-type (300 bp) and

KO alleles (422 bp) were resolved on a 2% agarose gel and visualized after ethidium bromide

staining.

+/+ +/- -/- +/+ +/- -/-

Serum Lung

SDS-PAGE

35 -

25 -

15 -

Western blotting

TN +/+ +/- -/- +/+ +/- -/-

Serum Lung

- TN

Genotyping

- KO PCR product (422 bp)- WT PCR product (300 bp)

100 -200 -300 -400 -500 -600 -

A

B

TN +/+ +/- -/-

bp

Fig. S2. Survey of TN protein abundance in various tissues. (A) Blood samples and various

tissues were harvested from normal healthy Balb/C mice and assayed for TN concentrations by

Western blotting analysis using a commercial rabbit mAb (Cat. No. ab108999, Abcam). SDS-

PAGE gel of corresponding tissue or serum proteins as sample loading controls for estimating

relative TN content per total protein. (B) Blood samples were collected from five normal healthy

Balb/C mice (7-8 weeks, 20-25 g), and serum TN content was estimated by Western blotting

analysis with reference to purified recombinant TN at various dilutions.

TN

/ P

rote

in

(AU

)

0

4

8

12

16

Bra

in

He

art

Kid

ne

y

Liv

er

Lu

ng

Se

rum

Sp

lee

n

35 -

25 -

15 -

- TN

Bra

in

He

art

Kid

ne

y

Liv

er

Lu

ng

Se

rum

Sp

lee

n

A SDS-PAGEWestern blotting

B

Se

rum

TN

(µg

/ml)

0

2

4

6

8

10

12 IQR

1.5 IQR

Median

Mean

Fig. S3. Parallel reduction of lung and serum TN content during lethal endotoxemia.

Balb/C mice were subjected to lethal endotoxemia and serum (A) and lung tissue (B) were

harvested at different time points after endotoxemia to measure TN content by Western blotting

analysis. n = 3 animals per group.

0 6 12 18 240

20

40

60

80

100

120

Lu

ng

TN

(%)

Time (h post LPS)

0 6 12 18 240

20

40

60

80

100

120S

eru

m T

N (

%)

Time (h post LPS)

A B

Fig. S4. Expression and purification of recombinant TN. (A) Amino acid sequence and

functional domains of human and murine TN. Three exons [1: 1-16; 2: 17-49, and 3: 50-181],

three predicted disulfide bonds (50-60, 77-176, and 152-168), and some key residues for each

functional domain are indicated. (B) Murine TN corresponding to amino acids 22-202 (or 1-181

excluding the 21-residue leader signal sequence) with a C-terminal histidine tag was expressed in

E. coli BL21 (DE3) pLysS cells and purified by histidine-affinity and Triton X-114 extraction to

remove contaminating endotoxins.

kDa

250 –

130 –

100 –

15 –

10 –

70 –

55 –

40 –

35 –

25 – - TN

+ D

TT

-D

TT

1 10 20 30 40 50 60 70 80 90 100

EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVCLKGTKVHMKCFLAFTQTKTFHEASEDCISRGGTLGTPQTGSENDALYEYL

|.||.|:||::|||||:|.:|||||||.|.|.||||||||||.|||||||||||||.:||.|||||.||||||||||||.||||||||:..||:||:||:

ESPTPKAKKAANAKKDLVSSKMFEELKNRMDVLAQEVALLKEKQALQTVCLKGTKVNLKCLLAFTQPKTFHEASEDCISQGGTLGTPQSELENEALFEYA

← Exon 1 →← Exon 2 →

50 60 77

176

101 110 120 130 140 150 160 170 180

RQSVGNEAEIWLGLNDMAAEGTWVDMTGARIAYKNWETEITAQPDGGKTENCAVLSGAANGKWFDKRCRDQLPYICQFGIV (human)

|.||||:|:||||||||||||.||||||:.|||||||||||.||||||.|||||||||||||||||||||||||||||:||

RHSVGNDANIWLGLNDMAAEGAWVDMTGGLLAYKNWETEITTQPDGGKAENCAALSGAANGKWFDKRCRDQLPYICQFAIV (murine)

Heparin Binding α-helix Trimerization

- - - S – S - - -

- - - - - - S – S - - - - - -

S – S

152 168

Carbohydrate

Recognition Domain, CRD

B

A Amino acid sequence of mature human and murine tetranectin proteins

(without the 21- amino acid leader signal sequence)

Fig. S5. Recombinant murine TN conferred a dose-dependent protection against lethal

sepsis. Balb/C mice were subjected to lethal sepsis, then intraperitoneally administered

recombinant murine TN at sub-physiological (0.1 mg/kg, A) or supra-physiological (1.0 mg/kg,

B) doses at indicated time points. Animal survival was monitored for two weeks. n = 9 - 10

animals for each experiment, but some experiments were repeated (N = 2; A) to ensure

reproducibility.

-1 0 1 2 3 4 13 140

2

4

6

8

10

Time (days post CLP)

# o

f S

urv

ivo

rs

TN (1.0 mg/kg)

Saline

22%

20%

(+2, +24 h)

-1 0 1 2 3 4 13 140

4

8

12

16

20

Time (days post CLP)

# o

f S

urv

ivo

rs

TN (0.1 mg/kg)

Saline

60%

35%

(+2, +24 h)

A B

~ ~~ ~

~ ~~ ~

~ ~~ ~

~ ~~ ~

Fig. S6. Epitope mapping and specificity of representative mAbs raised against

recombinant human TN. (A) Dot blotting analysis of polyclonal antibodies (“pAb”) and

monoclonal antibodies (“mAbs”) using synthetic peptides corresponding to sequences shown in

Fig. 4B. Most purified mAbs specifically recognized an epitope on a particular peptide, such as

P2, P5, or P7. (B) Normal healthy human (“H”) and murine (“M”) serum proteins were resolved

by SDS-PAGE, and Western blotted with the different mAbs. Most mAbs recognized a specific

band with the predicted molecular weight of TN in the serum of normal healthy human or mouse.

pAb mAb1, 3, 9

P2 P2

P5

mAb2, 5, 6, 8

P5

mAb10

P7

P6 P7

P10

A

H M

100 -

55 -

35 -25 -

15 -

10 -

kDa

130 -

170 -

70 -

SDS-PAGE

35 -

15 -

35 -

15 -

35 -

15 -

- TN35 -

15 -

-35 -

15 -

Dot blotting analysis of mAbs using synthetic peptides

B Western blotting analysis of mAbs using human and mouse serum

mAb2 mAb5 mAb6 mAb8 mAb9

H M H M H M H M H M

Fig. S7. Cross-reactivity and epitope mapping of a panel of P5-reacting mAbs. (A) Sequence

of ten P5-overlapping peptides used for antibody epitope mapping. (B) Epitope mapping of a

panel of three P5-reacting monoclonal antibodies.

23F6 (mAb5) 25B2 (mAb6)27B12 (mAb8)

P5-overlapping peptide sequencesA

B Epitope mapping of P5-reacting MAbs by peptide dot blot

Fig. S8. Epitope sequence homology between different mammalian species. (A) Mammals

sharing similar or identical epitope sequence for P5-5-reacting mAbs. (B) Mammalian species

sharing 100% amino acid sequence identity in the epitope sequence (NDALYEYLRQ) of their

respective TN proteins.

Baboon Bear Bovine Buffalo Camel Cattle Cougar

Elephant Goat Gorilla Hedgehog Horse Human Lemur

Monkey Pig Rabbit Rhinoceros Seal Sheep Tiger

Mammals sharing identical epitope sequence (NDALYEYLRQ) B

NEALFEYARQ (Rat, 70% identity, 100% similarity)

|:||:||:||

NEALFEYARH (Murine, 60% identity, 100% similarity)

|:||:||:|:

NDALYEYLRQ (Human)

||||||||||

NDALYEYLRQ (Other mammals listed below in Panel B, 100% identity)

Epitope sequence homology between human and other mammaliar speciesA

Fig. S9. Characteristics of a panel of human TN-specific mAbs. The isotype of each mAb was

determined using the Rapid ELISA Mouse Isotyping kit (Cat. # 37503, ThermoFisher Scientific)

as per the manufacturer’s instruction. The epitope and equilibrium dissociation constant (KD)

was determined as described in the materials and methods. There are three CDRs (CDR1, CDR2,

and CDR3) on each of the heavy and light chains that collectively formulate a pocket that comes

into contact with the antigen. The antigen contact structure was generated using on-line ProABC

Prediction of Antibody Contacts Software (http://circe.med.uniroma1.it/proABC/) after inputting

respective CDR sequences of each mAb.

mAb8

IgG2b

P5-5

2.02e-9

1.48e-8

Protection

mAb6

IgG1

P5-5

9.15e-10

1.00e-7

Protection

mAb5

IgG1

P5-5

8.03e-8

weak

No Effect

mAb2

IgG2a

P5

1.14e-10

2.31e-8

Protection

Name:

Isotype:

Epitope mapping:

KD for human TN:

KD for murine TN:

Effect on Sepsis:

Prediction of antigen

contact structure

(H-bond) :

Fig. S10. IgG isotype controls did not affect sepsis lethality. Balb/C mice were subjected to

lethal sepsis and then intraperitoneally administered various IgG isotype controls at the indicated

dose (2.0 mg/kg) and time points (24 and 48 h after CLP). Animals were monitored for two

weeks.

IgG Isotype Controls (2.0 mg/kg)

60%

66% Saline

IgG2b (irrelevant IgG26)

Time (h post CLP)

0

20

40

60

80

100

Su

rviv

al (%

)

1st2nd

0 20 40 60 80 336

~ ~~ ~

~ ~~ ~

70%

80% Saline

IgG1 (murine TN-non-reactive mAb5)

n = 10; P = 0.63

0

20

40

60

80

100

Su

rviv

al (%

)

1st2nd

0 20 40 60 80 336

~ ~~ ~

~ ~

70% Saline

n = 10; P = 0.98

IgG2a (irrelevant IgG19)70%

0

20

40

60

80

100

Su

rviv

al (%

)

1st2nd

0 20 40 60 80 336

~ ~~ ~~ ~

80%

77% Saline

IgG2b (anti-P7 mAb10)

n = 9-10; P = 0.91

n = 9-10; P = 0.76

0

20

40

60

80

100

Su

rviv

al (%

)

1st2nd

0 20 40 60 80 336

~ ~~ ~~ ~

Fig. S11. Distinct effects of P2- and P5-reacting mAbs on sepsis-induced TN depletion. Male

Balb/C mice were subjected to lethal sepsis, then intraperitoneally administered a P5-reacting

mAb8 (2.0 mg/kg) or a P2-reacting mAb9 (2.0 mg/kg) at 2 and 24 h after CLP. At 28 h after

CLP, animals were euthanized to harvest blood, and serum TN concentrations were determined

by Western blotting analysis. Equivalent sample loading was verified by SDS-PAGE analysis of

serum proteins in parallel.

kDa

250 –

100 –

15 –

70 –

55 –

40 –

35 –

25 –

10 –

N CLP

+

Veh

CLP

+

mAb8

CLP

+

mAb9

SDS-PAGE

- TN

25 –

15 –

Fig. S12. Divergent effects of mAb8 and mAb9 on sepsis-induced systemic KC

accumulation. Male Balb/C mice were subjected to lethal sepsis, then intraperitoneally

administered a P5-reacting mAb8 (2.0 mg/kg) or a P2-reacting mAb9 (2.0 mg/kg) at 2 and 24 h

after CLP. At 28 h after CLP, animals were euthanized to harvest blood, and serum

concentrations of 62 different cytokines and chemokines were determined by Cytokine Antibody

Arrays. KC is the murine homolog of human GRO.

+ CLP

SDFSDF--11aa

TARCTARC

TCATCA --33

TECKTECK

TIMPTIMP--11

TNFTNF

sTNFsTNF RIRI

sTNFsTNF RIIRII

TPOTPO

VCAMVCAM --11

VEGFVEGF --11

+ +

ILIL--44

ILIL--55

ILIL--66

ILIL--99

ILIL--1010

ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313

ILIL--1717

KCKC

LeptinLeptin RR

LeptinLeptin

LIXLIX

LL--selectinselectin

LPTLPT

MCP1MCP1

MCPMCP--55

MM--CSFCSF

MIGMIG

MIPMIP--11aa

MIPMIP--11gg

MIPMIP--22

MIPMIP--33bb

MIPMIP--33aa

PFPF-

-55

ILIL--66

ILIL--99

ILIL--1010

ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313

ILIL--1717

KCKC

LeptinLeptin RR

LeptinLeptin

LIXLIX

LL--selectinselectin

LPTLPT

MCP1MCP1

MCPMCP--55

MM--CSFCSF

MIGMIG

MIPMIP--11aa

MIPMIP--11gg

MIPMIP--22

MIPMIP--33bb

MIPMIP--33aa

PFPF--44

PP--SelectinSelectin

RANTESRANTES

SCFSCF

++

-

--

--

AxlAxl

BLCBLC

CD30 LCD30 L

CD30 TCD30 T

CD40CD40

CRGCRG--22

CTACKCTACK

CXCL16CXCL16

EotaxinEotaxin

EotaxinEotaxin--22

FasFas LL

FractalkineFractalkine

GCSFGCSF

GMGM --CSFCSF

IFNIFN--gg

IGFBPIGFBP--33

IGFBPIGFBP--55

IGFBPIGFBP--66

ILIL--11aa

ILIL--11bb

ILIL--22

ILIL--33

ILIL--3 3 RbRb

-

SDFSDF--11aa

TARCTARC

TCATCA --33

TECKTECK

TIMPTIMP--11

TNFTNF

sTNFsTNF RIRI

sTNFsTNF RIIRII

TPOTPO

VCAMVCAM --11

VEGFVEGF --11

+ +

ILIL--44

ILIL--55

ILIL--66

ILIL--99

ILIL--1010

ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313

ILIL--1717

KCKC

LeptinLeptin RR

LeptinLeptin

LIXLIX

LL--selectinselectin

LPTLPT

MCP1MCP1

MCPMCP--55

MM--CSFCSF

MIGMIG

MIPMIP--11aa

MIPMIP--11gg

MIPMIP--22

MIPMIP--33bb

MIPMIP--33aa

PFPF-

-55

ILIL--66

ILIL--99

ILIL--1010

ILIL--1212((p40/p70p40/p70))ILIL--1212((p70p70))ILIL--1313

ILIL--1717

KCKC

LeptinLeptin RR

LeptinLeptin

LIXLIX

LL--selectinselectin

LPTLPT

MCP1MCP1

MCPMCP--55

MM--CSFCSF

MIGMIG

MIPMIP--11aa

MIPMIP--11gg

MIPMIP--22

MIPMIP--33bb

MIPMIP--33aa

PFPF--44

PP--SelectinSelectin

RANTESRANTES

SCFSCF

+

-+

--

--

AxlAxl

BLCBLC

CD30 LCD30 L

CD30 TCD30 T

CD40CD40

CRGCRG--22

CTACKCTACK

CXCL16CXCL16

EotaxinEotaxin

EotaxinEotaxin--22

FasFas LL

FractalkineFractalkine

GCSFGCSF

GMGM --CSFCSF

IFNIFN--gg

IGFBPIGFBP--33

IGFBPIGFBP--55

IGFBPIGFBP--66

ILIL--11aa

ILIL--11bb

ILIL--22

ILIL--33

ILIL--3 3 RbRb

+

+

-

-

-

+

-

+

- CLP

+ CLP + Ab8 + CLP + Ab9

Fig. S13. TN enhanced HMGB1 uptake and possible degradation by macrophage cultures.

Macrophage cultures were incubated with HMGB1 in the absence or presence of TN or

Dynasore at indicated concentrations for 2 h, and cellular content of HMGB1 (A) or TN (B) was

determined by Western blotting analysis, with reference to housekeeping protein β-actin.

Increased cellular HMGB1 (A) or TN (B) content was associated with the appearance of lower

molecular bands (marked by empty arrowheads) that may be indicative of possible degradation.

The high molecular bands (marked by solid arrowheads) may indicate possible TN

oligomerization.

---

+--

++-HMGB1

TN

DYN

(0.5 mg/ml)

(10.0 mg/ml)

(8.0 mM)+

+

+

100 -70 -55 -

35 -25 -

15 -

10 -

kDa

130 -

250 -

- HMGB1

- β-actin - β-actin

- TN

100 -

55 -

35 -25 -

15 -

10 -

130 -

250 -

70 -

---

+--

++-HMGB1

TN

DYN

(0.5 mg/ml)

(10.0 mg/ml)

(8.0 mM)+

+

+

A B

Fig. S14. mAb8 and dynasore inhibited the TN/HMGB1-induced increase of trypan blue

dye uptake in macrophage cultures. Murine peritoneal macrophages were stimulated with TN

(10 µg/ml) or HMGB1 (0.5 µg/ml) either alone or together in the absence or presence of mAb8

(65 µg/ml) or dynasore (10.0 µM) for 16 h, then stained with trypan blue dye. Shown are

representative fields with trypan blue-stained cells (blue). Scale bars, 50 µm.

- Control + TN + HMGB1

+ TN + HMGB1 + TN + HMGB1 + mAb8 + TN + HMGB1 + Dyn

Fig. S15. Proposed model for the mAb8-mediated protection against lethal sepsis.

Endotoxemic and septic insults cause systemic accumulation of HMGB1 and concurrent

depletion of TN, partly because circulating HMGB1 binds to TN to facilitate the endocytosis and

possible degradation of TN/HMGB1 complexes. The endocytosis of TN/HMGB1 complex may

trigger ASC oligomerization and release, resulting in possible macrophage pyroptosis and

immunosuppression that may compromise effective pathogen elimination and contribute to

septic lethality. The P5-reactive mAbs, such as mAb8, could bind to TN to interrupt its

interaction with HMGB1, thereby impairing HMGB1 endocytosis and macrophage pyroptosis,

and possibly reversing the sepsis-induced immunosuppression to confer protection against lethal

sepsis.

Endotoxemia

Sepsis

HMGB1 TN

HMGB1 / TN

Pyroptosis

mAb8

Immunosuppression

Ineffective

Pathogen Elimination

Table S1. Reagent sources.

SOURCE IDENTIFIER

Antibodies

Mouse anti-β-actin antibody Sigma-Aldrich Cat. # A1978

Rabbit mAb against the C-terminal

region (residue 150 to the C-terminus)

of human TN

Abcam Cat. # ab108999

HRP conjugated donkey anti-rabbit IgG GE Healthcare Cat. # NA934

Rabbit polyclonal IgGs against murine

ASC

Santa Cruz

Biotechnology Cat. # sc-22514

Monoclonal IgG1 against human ASC Santa Cruz

Biotechnology Cat. # sc-271054

Chemicals, Peptides, and

Recombinant Proteins

Crude bacterial endotoxin

(lipopolysaccharide, LPS) Sigma-Aldrich E. coli 0111:B4

Human serum Sigma-Aldrich Cat. # H3667

Recombinant human SAA (also termed

Apo-SAA PeproTech Cat. # 300-13

Recombinant human TN ACROBiosystems Cat. # CLB-

H5226

Human macrophage colony-stimulating

factor (M-CSF) PeproTech Cat. # SRP-3110

Dulbecco’s modified Eagle medium

(DMEM)

Invitrogen/Life

Technologies Cat. # 11995-065

OPTI-MEM I Reduced-Serum Medium ThermoFisher Scientific Cat. # 31985062

Penicillin/streptomycin Invitrogen/Life

Technologies Cat. # 15140-122

Trypan blue Invitrogen/Life

Technologies Cat. # 15250-061

Alexa Fluor 555 labeling kit A ThermoFisher Scientific Cat. # A30007

Alexa Fluor 488 labeling kit A ThermoFisher Scientific Cat. # A30006

DAPI Vector Laboratories, Inc Cat. # H-1200

Chemicals, Peptides, and

Recombinant Proteins

Human CLEC3B/tetranectin ELISA kit RayBiotech Cat. # ELH-

CLEC3B-1

LDH Assay Kit Pointe Scientific Inc Cat. # L7572

Amine sensor chip Nicoya Lifesciences Cat. # SEN-Au-

100-10-AMINE

NTA sensor chip Nicoya Lifesciences Cat. # SEN-Au-

100-10-NTA

Murine Cytokine Antibody Arrays RayBiotech Inc Cat. #. M0308003

Human Cytokine Antibody C3 Arrays RayBiotech Inc Cat. # AAH-

CYT-3-4

AST assay kit Pointe Scientific Inc. Cat. # A7561

ALT assay kit Pointe Scientific Inc. Cat. # A7526

Mice

Balb/C mice Jackson Laboratory Stock # 000651

Heterozygous TN (“CLCE3B”)-KO

mice Jackson Laboratory Stock # 027554

Software and Algorithms

ProABC Prediction of Antibody

Contacts Software

http://circe.med.uniroma

1.it/proABC/

Table S2. Demographics of 44 normal healthy controls and 45 septic patients.

Normal Sepsis

Septic

Shock

Age Group

(14-30 )

(31-55)

(56-90)

(31-55)

(56-90)

(56-90)

Sample Size (n) 15

17 12 14 17 14

Age

Range 14 - 30

33 - 55 57 - 90 31 - 55 58 - 83 62 - 87

Mean

±

SD

21

±

5

45

±

8

66

±

9

42

±

11

71

±

8

72

±

8

Gender ratio

(M/F)

7/8

11/6

6/6

5/9

6/11

4/10

Plasma [TN]

Mean

±

SD

(SEM)

10.1

±

1.8

(0.5)

10.1

±

1.4

(0.3)

9.2

±

2.2

(0.6)

3.8

±

1.7

(0.4)

3.5

±

2.1

(0.5)

3.0

±

1.2

(0.3)