Divergent in vitro inflammatory responses to antibodies relevant to transfusion-related acute lung injury

John-Paul Tung, Wesley Bierman, Christine Knauth, Robert Flower, Melinda Dean BBTS Annual Meeting 2013

What is transfusion-related acute lung injury (TRALI)? • Severe and potentially fatal reaction

295 cases & 45 fatalities in UK from 1996 – 2012 1,2

172 fatalities in US from 2005 – 2012 3,4

133 TRALI cases in Australia from 2006 – June 2011 5

Considered under-diagnosed and under-reported 6 Mortality rate 5 – 10%7 (may be as high as 40% in ICU patients) 8

• Clinical diagnosis made based upon consensus definition 9: occurs during or within 6 hours of transfusion pulmonary oedema (bilateral infiltrates on CXR) hypoxemia (PaO2/FiO2 < 300 or SpO2 < 90%) no evidence of TACO (left atrial hypertension)

1. Chapman et al: Transfusion 2009 2. 2012 Annual SHOT Report 3. Fatalities reported to FDA following blood collection and transfusion- Annual summary for fiscal year 2007 4. “ “ - Annual summary for fiscal year 2012

5. Reesink et al: Vox Sang 2012 6. Kram et al: Eur J Anaesthesiol 2005 7. Silliman et al: Blood Rev 2009 8. Gajic et al: Respir Crit Care Med 2007 9. Kleinman et al: Transfusion 2004

TRALI pathogenesis: two-event mechanism IN

TRAV

ASC

ULA

R

Pulmonary endothelium

Fluid leakage

Y Antibody

Antigen activated neutrophil releasing reactive oxygen species & enzymes

resting endothelial cell resting neutrophil

primed neutrophil

EXTR

AVAS

CU

LAR

1st event

activated endothelial cell

Donation Y Y

Y Y Y

Donation

BRM

BRM

2nd event

BRM

Pulmonary oedema & hypoxemia TRALI

damaged endothelial cell

Rationale for study

• Different antibodies implicated: Anti-HNA Anti-HLA class I Anti-HLA class II

• Differences: Antigen distribution Not all antibodies are leucoagglutinating Not all antibodies prime neutrophil respiratory burst

In a whole blood transfusion model what are the inflammatory effects of antibodies targeting different

classes of TRALI relevant antigens? (i.e. HNA vs. HLA class I vs. HLA class II)

divergent mechanisms?

TRALI relevant antibodies investigated

Antibody Clone Antigen targeted IgG subtype

Stock conc.

(µg/mL)

Final conc.

(µg/mL) Supplier

HNA-2a MEM-166 CD177 Mouse IgG1 500 5 BD

HLA class I G46-2.6 HLA-A, -B, -C Mouse IgG1 500 5 BD

HLA class II Tu39 HLA-DR, -DP, -DQ Mouse IgG2a 500 5 BD

Isotype control N/A N/A Mouse IgG1 1,500 5 Santa

Cruz

• Future studies to utilise donor-derived antibodies e.g. FFP from donors with HNA-3a antibodies

Methods

Whole blood transfusion model • Assess monocyte and neutrophil specific inflammatory responses • Assess overall inflammatory response

Harvest culture

Whole blood transfusion model 37°C, 5%CO 1 hour

2

Permeabilise WBCs

RBC, red blood cell

+/ GolgiPlug

37°C, 5%CO 5 hours

2

Lyse RBCs

WBC, white blood cell

CBA analysis of culture supernatant

Intracellular staining

Media

LPS (0.23 μg/mL) 500 μL volunteer

whole blood

400 μL RPMI media

100 μL diluted Ab

With

G

olgi

Plu

g W

ithou

t G

olgi

Plu

g

Culture supernatant collected

Stain harvested cells CD45 (leucocytes) CD14 (monocytes) CD16 (neutrophils)

CBA, cytometric bead array

–

Cytokines / Chemokines IL-1α IL-1β IL-6 IL-8 IL-10 IL-12 TNF-α IFN-α MCP-1 IP-10 MIP-1α MIP-1β

Intracellular inflammatory mediator staining

Intracellular staining

IL-6 IL-1α MCP-1 IL-8 TNF-α IP-10 IL-10 MIP-1α IL-12 MIP-1β

Flow cytometry

CD

16 F

ITC

Untreated LPS

IL-1α PE

% change cf. no transfusion control

isotype

α-CD177

isotype

α-CD177 media

LPS

RESULTS Inflammatory effects of TRALI-relevant antibodies • anti-HNA-2a • anti-HLA class I • anti-HLA class II

% change cf. no transfusion control

Effect of anti-HNA-2a MoAb on inflammatory responses

ψ, cf. LPS + media-control

Media

LPS

2nd insult 1st insult

* ψ

* *

n=8. Paired t-test * p<0.05

Summary Without LPS No inflammatory response With LPS A modest pro-inflammatory response: ↑ neutrophil IL-8, ↑ overall

MIP-1α and MIP-1β

Neutrophil inflammatory response Overall inflammatory response

ψ cf. LPS + media-control, ξ, cf. media-only control

% change cf. no transfusion control

Effect of HLA class I MoAb on inflammatory responses

Media

LPS

2nd insult 1st insult

Media

LPS

Neutrophil IL-8

Neutrophil IL-10

pg/m

L

ξ **

* ψ

n=8. Paired t-test * p<0.05, ** p<0.01

pg/m

L

pg/m

L pg

/mL

Summary Without LPS Minimal immunosuppressive response: ↓ neutrophil IL-8 With LPS Predominantly pro-inflammatory response: ↓ neutrophil IL-10, ↑

overall MIP-1α, MIP-1β & IL-8, and ↓ MCP-1

Neutrophil inflammatory response Overall inflammatory response

Media

LPS

Effect of HLA class II MoAb on inflammatory responses

0

1000

2000

3000

0

50

100

150

0

500

1000

1500

0

20000

40000

60000

80000

100000

0

5000

10000

15000

20000

25000

0

500

1000

1500

IL-1β IL-8

MIP-1α MIP-1β

IP-10 TNF-α

pg/m

L pg

/mL

pg/m

L

media LPS media LPS

2nd insult 1st insult

Media

LPS

Media

LPS

Neutrophil inflammatory response

Monocyte inflammatory response

% change cf. no transfusion control

Overall inflammatory response

** *

* **

*** *** *

Effect of HLA class II MoAb on inflammatory responses

n=8. Paired t-test * p<0.05, ** p<0.01, *** p<0.001

Summary Without LPS No inflammatory response With LPS Pro-inflammatory response: ↑ neutrophil MIP-1β, ↑ monocyte TNF-α,

↓ monocyte IL-10, and ↑ overall IL-1β, IL-8, MIP-1α, MIP-1β, IP-10 & TNF-α

α-HNA-2a Without LPS No inflammatory response

α-HLA class I Without LPS Minimal immunosuppressive response: ↓ neutrophil IL-8

α-HLA class II Without LPS No inflammatory response

Results summary

α-HNA-2a Without LPS No inflammatory response With LPS A modest pro-inflammatory response: ↑ neutrophil IL-8, ↑ overall

MIP-1α and MIP-1β α-HLA class I Without LPS Minimal immunosuppressive response: ↓ neutrophil IL-8 With LPS Predominantly pro-inflammatory response: ↓ neutrophil IL-10, ↑

overall MIP-1α, MIP-1β & IL-8, and ↓ MCP-1 α-HLA class II Without LPS No inflammatory response With LPS Pro-inflammatory response: ↑ neutrophil MIP-1β, ↑ monocyte TNF-α,

↓ monocyte IL-10, and ↑ overall IL-1β, IL-8, MIP-1α, MIP-1β, IP-10 & TNF-α

Results summary

Conclusions

• Minimal immunomodulation in the absence of LPS Importance of patient factors as a 1st event in TRALI

pathogenesis

• In the presence of LPS, treatment with HNA-2a, HLA class I and HLA class II monoclonal antibodies each elicited distinct pro-inflammatory profiles

Evidence of divergent pathophysiological mechanisms in antibody mediated TRALI pathogenesis

Acknowledgements & Further Information

Australian governments fully fund Red Cross for the provision of blood products and services to the Australian community

• Volunteer blood donors • BBTS

• John-Paul Tung jtung@redcrossblood.org.au (07) 3838 9146