Mucosal Associated Invariant T-cells

Olivier Lantz, Inserm U932, Institut Curie, Paris, France

NK T and MAIT cells: Two reciprocal populations in mice and humans?

B Cell

(Bendelac et al, Ann Rev Immunol, 2007; Treiner and Lantz, Current Opin Immunol, 2006)

mouse human mouse human

iTCR V a Chain Va14-Ja18 Va24-Ja18 Va19-Ja33 Va7.2-Ja33/20/12

Associated Vb Vb2,7 and 8 Vb11 Vb8 and Vb6Vb13, 2, 22 and

others

Co-receptor

Abundance1 % spleen

20-40% liver

0.01-0.5% blood

1% liver0.01-0.5%

1-10% blood

20-40% liver

Location

Restr iction

Antigène

Selecting cells

Per ipheral requirements

CD1d MR1

liver, thymus, spleen Gut and lung LP, liver, blood

IL-15 commensal flora and B lymphocytes

NKT M AI T

DN, few CD4

DP cortical thymocytes DP cortical thymocytes

CD8ab, DN and CD8aa

5-AR-U derivatives (Riboflavin

precursor)Glycolipids

Bacterial activation of MAIT cells

CD8

CD

4

Ant-MR1

Iso Ctl

Va7.2

CD

16

1

Human MAIT

(Va7.2 Pos fraction) CD8+DN

CD69

+ E. Coli

CD69

CD8

DN

CD4

MR1+ MR1-

TCR

V

6

CD8

CD

4

Mice: iVa19/V6 Tg

MAIT cells respond to APCs infected with bacteria in a MR1

dependent manner both in human and in mouse Le Bourhis et al, Nature Immunol. 2010

Activation of iVa19V6-Tg MAIT cells by BCG infected BMDCs

SS

C-A

SS

C-A

SS

C-A

CD

19

CD

4

CD

25

Vb6

TCRb

Spleen

MLN

iVa19V6 Tg Ca-/-

MR1+ or MR1-

MR1+ or MR1-

BM

CD11b/CD11c/CD19

depletion

+ GM-CSF

(20ng/ml)

>5 days

+ BCG (or E.coli)

1h

BMDC

ON co-culture

Va19V6Tg MR1+ DC MR1+

--------------- MR1neg --------

--------------- MR1+ DC MR1neg

--------------- MR1neg ---------

0

20

40

60

80

100

0 1 10 100

E. coli MOI

CD

69

+CD

25

+ (%

)

0

10

20

30

40

50

0 3 10 30

BCG MOI

CD8+ DN Va19Vb6Tg T cells + BMDC

Activation of iVa19-V6-Tg MAIT cells by BCG infected BMDCs

Bacteria

Gram-

E. coli +

K. pneumoniae +

P. aeroginosa +

S. flexneri +

S. paratyphi A +

H. influenzea +

Gram+

L. monocytogenes -

S. epidermidis +

S. aureus, +

S. group A -

E. faecalis -

Other M. abscesus +

Yeasts

S. cerevisae +

C. glabrata +

C. albicans +

Fungi A. Fumigatus

(Conidia) -

Parasites Schistosome

(eggs) -

Viruses

EMCV -

Para-influenzae -

HSV -

Sendai -

NDV -

?

?

Rib neg

Rib neg

GTP

RL-6-Me-7-OH

Hydrogens are not displayed

Ribulose 5’ phosphate

RibA RibD

?

Phosphorylated 5-A-RU

5-A-RU

RL-6,7-diMe

3,4 Dihydroxy-2-butanone-4 Phosphate

Riboflavin

Non-enzymatic reactions

Methylglyoxal

or

Glyoxal

Pyrimidine adducts

+

5-OP-RU

RibB

Lumazine synthase (RibE)

5-OE-RU

J. McCluskey group, Nature, 2014

C. Soudais et al, JI, 2015

Testing gram- bacteria Rib mutants and mouse MAITs

Va19-Tg

WT3mMR1

C. SOUDAIS, JI, 2015

ribA

ribB ribE

RibD

CD

25

+ C

D69

+ c

ells

(%

)

E coli

A

E

Rib bacterial

mutant

B

D

MOI

5-A-RU is the MAIT ligand precursor

MAIT ligands

• 5-A-RU is necessary for MAIT activation by both gram+ and gram-

bacteria

• This is true for both human and mouse MAITs

• 5-A-RU + Methyl glyoxal are sufficient to activate most iVa19 Tg MAIT

cells (either V6 or V8) in vitro and in vivo.

• Little indication for bacterial ligand heterogeneity in 3 model bacteria (E.

coli, S. typhimurium, L. lactis)

• Of non-bacterial origin?

• Pharmacological agonists or antagonists.

MAIT and mates

5-OP-RU loaded mouse MR1 tets stain human MAIT cells

CD8a

CD4

CD3+

Tet+

J. Altman

B6 CAST F1 N20.1

1

10

100 MLN

Va

19-J

a33

rela

tive e

xp

ressio

n B6/Cast (F1)

B6/B6 (B6)

The Castaneus strain harbors 20 fold MAIT cells than

C57Bl/6 mice. Linked to one locus

Generation of a B6-MAITCast congenic strain

Y. Cui, K. Franciskiewicz, JCI 2015

CD4 CD8 DN

CD44

RORγt-

GF

P

MR1+

MR1-

MR1-dependent DN GFP+ T cells in RORgt-GFPTG B6-MAITCast strain

Gated on TCRβ+ CD90.2+ CD1d-α-GC tetramerneg

Lung

CD4 CD8 DN NKT0.01

0.1

1

10

% R

OR

c(γ

t)+

of to

tal T

cells

Liver

CD4 CD8 DN10-2

10-1

100

101

102

% o

f R

OR

c(g

t)+ T

cell

MR1+ MR1-

Y. Cui, K. Franciskiewicz, JCI 2015

Phenotype of MAIT cells in mice

Spleen

Murine MAIT cells are similar to human MAIT and express PLZF

Y. Cui, K. Franciskiewicz, JCI 2015

Th1/2/10/17 secretion pattern of murine MAIT

cells

FACS sorted GFP+ DNCD8lo T cells from RorgtgfpTG B6-MAITCAST mice activated by anti-CD3+CD28 beads 48h

E.coli MOI = 1 E.coli MOI = 10 PMA/Iono

The cytokine production by unpurified murine MAIT cells

is very variable according to the method of stimulation (1)

0

anti-CD3/28

PMA/Ionomycin 5h

MOI=0.1

MOI=1

MOI=10

9h

The cytokine production by unpurified murine MAIT cells

is very variable according to the method of stimulation (2)

0

anti-CD3/28

MOI=0.001

MOI=0.01

MOI=0.1

MOI=1

Overnight

The cytokine production by unpurified murine MAIT cells

is very variable according to the method of stimulation (3)

Purified murine MAIT cells are activated by a semi-purified

bacterial fraction in an MR1 dependent manner

Y. Cui, K. Franciskiewicz, JCI 2015

DN CD8lo

RORgt-GFP+

0 0.1 1 10 0

20

40

60

80

100 BMDC MR1+

α-MR1

α-IL12

BMDC MR1-

α-MR1

α-IL12

MOI

% C

D25

+ C

D69

+

Unpurified mouse MAITs cells are activated by soluble

factors

Anti-MR1

Anti-IL12

Yue Cui, unpublished

CD44

Memory

NKG2D

NK Receptor

CD4−CD8−

Coreceptor

IL-18Rα IL-12Rβ

IL-7Rα

Cytokine Receptor

CD103

CCR6

Tissue homing

CXCR6

iTCRα

mVα19-Jα33

MR1

5-A-RU

metabolites

PLZF

RORγt

Cytokine

IFN- IL-17

IL-4, IL-10 Mouse: 1-2% in liver

2-3% in lung

0.1-0.2% in spleen

21 Y. Cui, K. Franciskiewicz, JCI 2015

Intravesical instillation of

UTI89 (107 CFU/mouse)

days 0 7 2

Bladder enzymatic dissociation

(Liberase, collagenase D, DNase)

CFU evaluation

FACS analysis

-1

20H UTI89* static culture

OD

60

0

MR1+ or MR1-

* UTI89: uropathogenic E. coli strain (UPEC) isolated from a patient with an acute bladder infection.

Experimental urinary tract infection

Recruitment of lymphocytes to the bladder of UTI89 infected mice

CD8 C

D4

MR1+

MR1-/-

MR1+

MR1-/-

MR1+

MR1-/-

Day 2 p.i.

Day 7 p.i.

Day 0

TCR+ T cells

36

59 0.5

43

49 1.2

37 0.6

62

15 0.8

84

59 0.7

39

35 0.5

63

T cells

CD4 T cells

Efficient clearance of UTI89 is correlated with

increased bladder-infiltrating MAIT cells

CD8 C

D4

MR1+

MR1-/-

MR1+

MR1-/-

MR1+

MR1-/-

Day 2 p.i.

Day 7 p.i.

Day 0

TCR+ T cells DN

36

59 0.5

43

49 1.2

37 0.6

62

15 0.8

84

59 0.7

39

35 0.5

63

62

(80)

26

(16)

53

(306)

26

(118)

43

(400)

3

(154)

CD44

RO

R

t-G

FP

MAIT cells

Anti-microbial activity of murine MAIT cells

• Context dependent effector activities of MAIT cells like in humans

• Protection in TCRa and TCR transgenic models

• Migration to the site of infections as in humans

• The effect in polyclonal models is more difficult to evidence

• Could be related to the still rather low number of MAIT cells even in the B6-MAITcast model: why?

MAIT expand much less in mice than in humans

Yvonne Mburu, Stanislas Mondot

Repertoire analysis by NGS

MiSeq

Human samples (5'RACE)

Mouse samples V8 primer

Differences:

• Mouse vs human physiology?

• Study of tissue vs blood MAIT cells

• Microbial exposures (early life)?

- microbiota

- pathogens

Low frequencies of MAIT in mice correlated with lower expansion in comparison with humans: why?

Decreased MAIT numbers in subjects with high

infection burden (Zimbabwe)

% o

f in

dic

ated

su

bse

t in

gd

neg

CD

3+

MAIT NKT

Genetic or environment? F. Mutapi, N. Naush, Edimburg U

% of MAIT (T cells)

- E.coli

1

10

100

CD

8lo

DN

/CD

44

+/G

FP+

%

of

T ce

lls

CD69

CD

25

CD44

GF

P

CD

69

+CD

25

+ (%

)

+E.coli

LIVER

0 18h

i.v. injection of:

- E.coli (DH5a) 10^9 CD1dKO RORγt-GFPTG B6-MAITCast/Cast

Accumulation of MAIT cells in the liver upon systemic infection with E.coli

0

21

MAIT quantification in the liver

i.v. injection of:

- E.coli (DH5a)

10^7

B6-MAITCast/Cast RORγt-GFPTg

MAIT activation and expansion upon systemic infection with E.coli

days

28

28

rechallenge:

- E.coli (DH5a)

10^7

NKT %

- 28DAYS 21+7DAYS

0

25

50

TCRb

CD

1d

-TT

m

Gate: CD19- TCRβ+

DAY 28 DAY 21+7

% o

f T

CR

b+

CD

19

-

NKT

DAY 28 DAY 21+7 p.i.

n.i.

n.i.

Decreased number of NKT cells after systemic E. Coli infection

K Franciszkiewicz et al unpublished

CD44

GF

P

Gate: CD8loDN TCRβ+ T cells

DAY 28 DAY 21+7

MAIT (GFP+ DN CD8lo)

DAY 28 DAY 21+7 p.i.

n.i.

n.i.

Increased number of MAIT cells after systemic E. Coli infection

MAIT #

- 28DAYS 21+7DAYS

1.0x103

1.0x104

1.0x105

1.0x106

DAY 28 DAY 21+7 p.i. n.i. Cell counts per liver

Probable loss of RORgt expression by MAIT cells after bacterial rechallenge

Improving the B6-MAITcast model

• Systemic infection with a non pathogenic E. Coli strain increases the number of MAIT cells in the liver

• Work in progress

• Hampered by the unvailability of murine 5-OP-RU-MR-1 tetramers

• Analysis of anti-bacterial activity or other functions of MAIT cells in vivo

Acknowledgments Institut Curie / INSERM U932

Katarzyna Franciszkiewicz

Marion Salou

François Legoux

Qian Zhou

Anna Schneider

Aurélie Darbois-Delahousse

Virginie Premel

Stanislas Mondot

Alexandra Kachaner

Yue Cui

Claire Soudais

Manal Sarkis

Yvonne Mburu

Stephanie Bessoles

Lionel Le Bourhis

Natalie Seach

Collaborators

Susan Gilfillan

Ted Hansen

Shou Huang

J. Altman

S. Caillat-Zucman

• MR1 the most conserved MHC1b molecule

• The Va segment used by MAIT cells is

- The most conserved

- One member family in all sequenced species despite a

number of Va segments varying from 45 to 350

- The most 5’ in all species: the latest to rearrange

• The Ja33 segment is the most conserved

Striking phylogenetic conservation of both the MAIT TCRa

segments and MR1 molecule in mammalians and marsupials

Lopez-Sagaseta et al, PNAS, 2013

MR1 is the most evolutionarily conserved MHC-I molecule

P. Boudinot et al submitted

MR1

PLZF

Ror(gt)

G0 (KI-

67neg)

Tissue homing CCR6

CXCR6

CCR9

iTCRa

iVah7.2 iVam19-Ja33

Vh2/13/22

Vm6/8

CD8aint, CD8aa or DN

CD161

NKp30 NK Receptors

IL-18R

IL-12R

IL-23R

CD45RO

CD26

Memory

IL-2R Riboflavin

Derivatives

MAIT cell phenotype

MDR Tilloy, JEM, 1999

Treiner, Nature, 2003

Martin, PLoS Bio, 2009

Le Bourhis, Nat imm, 2010

Dusseaux, Blood, 2011

Le Bourhisl, COI, 2013

Le Bourhis, PLoS Path, 2013

Seach, JI, 2013

Soudais, JI, 2015

Cui, Franciszkiewicz, JCI, 2015

MAIT cells represent 1 to 8% of blood T lymphocytes

20-40 of liver lymphocytes

spleen

6 13 17 24 3010-1

100

101

102

103MR1+MR1-

Age (d)

Va

19-J

a33

rela

tive e

xpre

ssio

n

liver

6 13 17 24 3010-1

100

101

102

103MR1+MR1-

Age (d)

Va

19-J

a33

rela

tive e

xpre

ssio

n

Mouse MAIT cells home to liver after birth

thymus

6 13 17 24 3010-1

100

101

102

103MR1+MR1-

Age (d)

Va

19-J

a33

rela

tive e

xpre

ssio

n

39

Microbial 5-A-RU + metabolites from the intermediary metabolism activate

mouse MAIT cells

E coli fraction

5-A-RU

5-A-RU/ DHA

5-A-RU/ MetG

5-N-RU ± DHA or MetG

CD

25

+ C

D6

9+

ce

lls (

%)

Chemicals, mM

iVa19 Tg

Methylglyoxal

(MetG)

Di-hydroxy acetone

(DHA)

or

5-Amino-6-D-

ribitylaminouracil

(5-A-RU)

5-Nitro-6-D-

ribitylaminouracil

(5-N-RU)

C. SOUDAIS, JI, 2015

Activation of MAIT cells