At the crossroads of immunology and atherosclerosis ... · • Mach F, et al. Nature. 1998.; 394:200-203 • Differentiation into the Th1 cell subset is promoted by antigen presentation

At the crossroads of

immunology and atherosclerosis

– clinical implications

Roland Klingenberg, MD, FESC

Conflict of Interest - Disclosures

There are no financial interests to report within the past 12 months that

may pose a potential conflicts of interest.

Current employer: Kerckhoff-Klinik, Bad Nauheim, Germany

Diversity of immune cells in atherosclerosis

CD4+ T helper cell subsets

Th17 cells – dual role

Th1 cells – pro-atherogenic

Th2 cells – dual role

Treg cells – anti-atherogenic

Modified from Klingenberg R, Hansson GK. Eur Heart J. 2009; 30:2838-2844

Comprehensive update in Ait-Oufella H et al. Circ Res. 2014;114:1640-1660

• Major T cell subset in atherosclerotic lesions • Frostegard J, et al. Atherosclerosis. 1999; 145: 33-43

• Bona fide transcription factor T-bet and signature cytokine IFN-

• Drives arterial inflammation and promotes lesion progression • Szabo SJ, et al. 2002. Science. 2002; 295:338-342

• Buono C, et al. Proc Natl Acad Sci U S A. 2005; 102:1596-1601

• Gupta S, et al. J Clin Invest. 1997; 99:2752-2761

• Signalling via the CD40 costimulation pathway is essential for

the development of atherosclerotic lesions • Mach F, et al. Nature. 1998.; 394:200-203

• Differentiation into the Th1 cell subset is promoted by antigen

presentation through APC in the context of a CD40-CD40L

costimulatory signal• Lutgens E et al. J Exp Med. 2010; 207:391-404.

CD4+ Th1 cell subset in atherosclerosis

Regulatory T cells come in distinct flavors

modified from Shevach EM. Immunity. 2006; 25:195-201

CD4+ CD25+ natural Treg- Ait-Oufella H, et al.

Nature Medicine. 2006; 12:178–180

- Mor A, et al.

ATVB. 2007; 27:893-900

BUT: CAUSALITY (Foxp3) ?

MECHANISM OF ACTION ?

Klingenberg R, et al.

JCI 2013; 123:1323-1334

Natural Antigen-induced Treg cells in atherosclerosis

Transcription factor FoxP3:

key lineage marker and master switch in regulation and development

Fontenot, JD, et al. Nat Immunol. 2003; 4:330-336

Hori, S, et al. Science. 2003; 299:1057-1061

Khattri, R, et al. Nat Immunol. 2003; 4:337-342

Depletion of transgenic Treg cells propagates atherosclerosis

Klingenberg R, et al. J Clin Invest. 2013; 123:1323-1334

Increased necrotic core with few cells but no increase in vascular inflammation

Klingenberg R, et al. J Clin Invest. 2013; 123:1323-1334

Depletion of Treg cells promotes hypercholesterolemia (VLDL/CMR fraction)

Klingenberg R, et al. J Clin Invest. 2013; 123:1323-1334

Impaired VLDL/CMR lipoprotein clearance upon Treg cell-depletion

Klingenberg R, et al. J Clin Invest. 2013; 123:1323-1334

Increased activity of LPL, HL and PLTP, decreased Sort1 protein

Klingenberg R, et al. J Clin Invest. 2013; 123:1323-1334

Plaque regression upon natural Treg expansion by anti-CD3 Ab in ldlr−/− mice

Modified from Kita T et al. Cardiovasc Res. 2014; 102:107-117

1) Administration of anti-CD3 Ab mediated:

- Increase in Treg cells in spleen, lymph node and

atherosclerotic lesion

- Decrease in macrophages in atherosclerotic lesion

- Increase in collagen in atherosclerotic lesion

2) Depletion of Treg cells by co-administration of anti-CD25Ab:

- Abrogated effect of anti-CD3 Ab on lesion regression

Increased infarct size in Treg-cell–ablated Foxp3DTR mice compared with WT

Weirather J, et al. Circulation Research. 2014; 115:55-67

Expansion of natural Treg cells improves survival and reduces LV ruptures

Weirather J, et al. Circulation Research. 2014; 115:55-67

Diversity of regulatory T cells in atherosclerosis

modified from Shevach EM. Immunity. 2006; 25:195-201

Wigren M, et al.

J Intern Med. 2012; 269:546-56

Herbin O, et al.

ATVB. 2012; 32:605-612

Klingenberg R, et al.

ATVB 2010; 30:946-952

Klingenberg R, et al.

JCI 2013; 123:1323-1334

Natural Antigen-induced Treg cells by vaccination

All used apoB-100 peptide p210 as vaccine antigen

BUT: different route and adjuvants effecting distinct mechanism of action

Intranasal p210-CTB reduces atherosclerosis in apoe−/− mice

Klingenberg R, et al. ATVB. 2010; 30:946-952

Increased IL-10+ Tr1 (Treg) cells and apoB-100-specific Treg cell activity in spleen

Klingenberg R, et al. ATVB. 2010; 30:946-952

Human setting – Treg cells in clinical atherosclerosis

Modified from Klingenberg R, Hansson GK.

Eur Heart J. 2009; 30:2838-2844

Clinical data are scarce:

de Boer OJ et al. Low numbers of FOXP3 positive regulatory T

cells are present in all developmental stages of human

atherosclerotic lesions. PLoS One. 2007; 2:e779.

Mor A., et al. Altered status of CD4(+)CD25(+) regulatory T cells

in patients with acute coronary syndromes. Eur Heart J. 2006;

27:2530-2537.

Han, SF, et al. The opposite-direction modulation of CD4+CD25+

Tregs and T helper 1 cells in acute coronary syndromes. Clin

Immunol. 2007; 124:90-97.

Cheng X, et al. The Th17/Treg imbalance in patients with acute

coronary syndrome. Clin Immunol. 2008; 127:89-97.

Ammirati E, et al. Circulating CD4+CD25hiCD127lo regulatory T-

Cell levels do not reflect the extent or severity of carotid and

coronary atherosclerosis. ATVB. 2010; 30:1832-1841.

Wigren M, et al. Low Levels of Circulating CD4+FoxP3+ T Cells

Are Associated With an Increased Risk for Development of

Myocardial Infarction But Not for Stroke. ATVB. 2012; 32:2000-

2004.

Thrombus and blood to analyze T cell subsets and Antigen-specificity in ACS

FACS analysis

DNA analysis

V-J gene segments encode variable chains

one cell = one rearrangement

Splicing

Vn - Ji AC

Vn – Ji AC

Env. 10Kb

V genes J genes AC

Identification of V-J rearrangements at the genomic level

276 gene segment combinations possible = 100%

Analysis: ImmunTraCkeR® PCR technology

+ peripheral blood

Thrombus

Predominance of Treg cells in coronary thrombi vs. peripheral blood in ACS

Klingenberg R et al. Eur Heart J. 2015; 36:1041-8

Reduced TCR diversity in coronary thrombus vs. peripheral blood in ACS

Klingenberg R et al. Eur Heart J. 2015; 36:1041-8

Intra-individual comparisons of TCR diversity

expressed as percentage of 276 possible

V(D)J gene segment rearrangements in the

human TCR β chain (hTRB)

Reduced TCR diversity in peripheral blood (ACS vs. healthy)

Klingenberg R et al. Eur Heart J. 2015; 36:1041-8

Approaches how to enhance inherent atheroprotective immunity (Treg cells)

Modified from Klingenberg R, Hansson GK. Eur Heart J. 2009; 30:2838-2844

Expansion of Treg cells in vivo:

CLINICAL

- Sirolimus (Peccatori J, et al. Leukemia 2015;29:396-405)

- IL-2 (Saadoun D, et al. NEJM 2011;365:2067-2077)

EXPERIMENTAL

- Anti-CD3 antibody therapy

- Modulation of co-stimulatory molecules (ICOS,

PD-1, OX40L, and CD137)

- Cytokine administration (IL-10, IL-2)

- Calcitriol (Vitamin D3)

- Viral proteins (Measles virus, BCG)

- Vaccination (antigen-specific)

Expansion ex vivo and adoptive transfer

Antigen-specific expansion in vivo:

- vaccination

Controlled Level Everolimus in Acute

Coronary Syndromes (CLEVER-ACS)

NCT01529554

Göran Hansson, Thomas F. Lüscher,

Nobert Gerdes, Christian M. Matter,

Anton Gistera, Daniel F. Ketelhuth, Anna M. Lundberg,

Mats Rudling, Stefan K. Nilsson, Gunilla Olivecrona,

Stefan Zoller, Robert M. Badeau, Daniela Strodthoff,

Michael Lebens, Andreas Hermansson,

Gunilla N Fredrikson, Christine Lohmann,

Chad E. Brokopp, Audrey Grivès, Anais Courtier,

Milosz Jaguszewski, Nicolas Pasqual,

Eugenia Vlaskou Badra, Anika Lewandowski,

Oliver Gämperli, Simon P. Hoerstrup, Willibald Maier,

Ulf Landmesser, Jan Holmgren, Jan Nilsson, Matti

Jauhiainen, Tim Sparwasser