Evolution of anti-donor alloimmunity following transplantation -

implications for CAN

Maria Hernandez-FuentesRichard BakerWan Fai Ng

Osquel Barroso-Herrera

Indirect allorecognitionIndirect allorecognition

IL-2IL-2

II

IIII

Allorecognition: the two pathways

DirectDirect allorecognition allorecognition

allo APC

Shed allogeneic MHC

CD8 CD4

Auto APC

Taken up and processed by auto APC IIII

IL-2IL-2

II

CD8 CD4

allo APC

HTLp IL-2HTLp IL-2

CD45RACD45RA++ (Naïve) and CD45RO (Naïve) and CD45RO++ (Memory) T cells (Memory) T cellshave different patterns of recirculation. have different patterns of recirculation.

THYMUSTHYMUS

BLOODSTREAMBLOODSTREAM

HEVHEVLYMPHNODE

THORACIC DUCT

tissue tissue parenchymparenchym

al cell al cell

““RA circuit”RA circuit”

““RO circuit”RO circuit”

A significant drop in the anti-donor A significant drop in the anti-donor response is only observed in the response is only observed in the

CD4CD4++CD45ROCD45RO++ fraction. fraction.

p>0.05(Wilcoxon)

Ratio of frequency post/pre for CD4+CD45RA+ cells

TH LM AT AL DM JW SA CS SAl

Ratio

10-3

10-2

10-1

100

101

102

103

RA donor RA 3rd party

Ratio of frequency post/pre for CD4+CD45RO+ cells

TH LM AT AL DM JW SA CS SAlR

atio10-3

10-2

10-1

100

101

102

103

p=0.008(Wilcoxon)

RO donor RO 3rd party

33rdrd party ratios close to unity mean that immunosuppression has party ratios close to unity mean that immunosuppression has not significantly affected the lymphocyte response; as can be not significantly affected the lymphocyte response; as can be observed for both fractions.observed for both fractions.

deletion anergy

Mechanisms of peripheral toleranceMechanisms of peripheral tolerance

deletion anergy

regulation

?

ignorance

Experimental Design

+ IL2 (30 U/ml) for 3 days

Wash and rest for 1 day

CD4 cells

LDA

LDA

PBMCadherenceon plastic

1 hrCocktails of mAbFollowed by magnetic beads to remove non-CD4 cells

Readout:

IL2 bioassay with CTLL

Anti-donor frequencies before and after IL-2T.H

.

J.W

.

J.B

.

A.W

.

M.M

.

R.D

.

S.E.

C.S.

K.M

.

P.K

.

G.K

.

D.T

.

100

1000

10000

100000

1000000

1/Freq

Non Hypo-responsive PatientsDonor Specific Hypo-responsive Patients

Patients with Reveral of Hyporesponsiveness in bold

Post IL-2

Pre IL-2

Donor-specific hyporesponsiveness was reversed in Donor-specific hyporesponsiveness was reversed in 5 out of 5 patients with IL-2 5 out of 5 patients with IL-2

Elevated frequencies of T cells with indirect anti-donor

allospecificity in patients with chronic transplant rejection

Vella et al, ‘97 Transplantation renal transplant recipients

Ciubotariu et al, ‘98 JCI heart transplant recipients

Hornick et al, ‘00 Circulation .. .. ..

SivaSai et al, ‘99 Transplantation lung transplant recipients

Baker et al, 2001 JI renal transplant recipients

1/f

req

ue

ncy

104

105

106

p<0.05 *

CAN CAN Free

Lymph Node

Direct pathway

Indirect pathway

Mechanisms of peripheral toleranceMechanisms of peripheral tolerance

deletion anergy

regulation

?

ignorance

Direct pathway naïve T cells?

Direct pathway - memory T cells

Evolution of anti-donor direct and indirect alloresponses following transplantation using conventional immunosuppression

Indirect pathway T cells - in some patients

B

AB

Graft acceptance

Tolerant to AA

The T cells that transfer tolerance appear to have indirect indirect allospecificity

CD4+

Tolerance to MHC class I-only mismatched graft transferred by CD4 T cells (Waldmann)

Pre-treatment with donor MHC class I induces tolerance when combined with anti-CD4 mAb(Wood)

Tolerance could not be induced unless the indirect pathway was intact (Auchincloss)

“Indirect recognition by helper T cells can induce donor-specific cytotoxic T lymphocytes in vivo”

RS Lee, ……. H Auchincloss Jr.

J. Exp. Med. 1994 179:865

CD4

Immature recipient DC

Mature recipient DC presenting donor MHC indirectly

Donor parenchymal cells

Unlinked help and suppression - the “4 cell problem”

Unlinked help or suppression…?

Lymph Node

CD8

CFSE-R1

R1 x R4Acceptor DC only

R1

R4

0h

20h

0h

20h3.5%13.8%48.5%

2.9%2.8%3.0%

0.1%1.1%0.5%

M1

2.5%3.7%2.3%

M1

0.4%1.5%0.8%

3.7%10.1%41.2%

4.5%13.7%19.4%

1.8%15.4%23.1%

H-2Ab

immatureBALB/c DCs

matureBALB/c DCs

H-2Kb

C57BL/6DCs

Co-culture of MHC-mismatched DC leads to class I and II exchange

1.3%7.6%7.5%

1.6%23.9%17.4%

1.2%19.0%18.3%

1.7%5.0%7.2%

H-2AbH-2Kb

MHC transfer between DCs can occur in the absence of cell contact

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

S t i m u l a t o r c e l l s

T : D C = 1 0 : 1

CPM

0

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

S t i m u l a t o r c e l l s

T : D C = 1 0 : 1

Responders:BALB/c CD4+ T cells

Responders:BALB/c CD8+ T cells

Stimulators:

BALB/c DCs alone.

BALB/c DCs from mixe d cultures .

C57BL/6 DC s-CFS E from mixed culture s .

BALB/c DCs from trans -well cultures .

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 : 1 5 : 1 1 0 : 1

T : D C r a t i o

CPM

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 : 1 5 : 1 1 0 : 1

T : D C r a t i o

Respo nder s:F5 CD8+ T ce lls

Respo nder s:OT-II CD4+ T ce lls

Stimula tors:

o BALB/c DCs alone.

æBALB/c DCs from mixed cultures.

= C57BL/6 DCs-CFSE from mixed cultures.

◊ BALB/c DCs from trans-well cultures.

99.9%

99.5%

Mixed cultureCFSE+veC57BL/6 DCs

CFSE-veBALB/c DCs

FSC

FSC

FSC

Transferred MHC:peptide complexes are recognised by T cells

0.260.5 37.5

1.8

4.360.3 4.4

34.9

0.157.5 29.8

2.6

4.856.8 3.2

35.2

0.242.1 57.2

0.5 0.142.2 57.4

0.3

CFSE

ImmatureBALB/c DCs

MatureBALB/c DCs

(CBA ECs+ IFNγ) -CFSE

C57BL/6 ECs -CFSE

(C57BL/6 ECs+ IFNγ) -CFSE

DCs acquire MHC class I from γ-IFN-treated ECs

1.020.4 25.1

53.5 0.620.7 22.7

56.0

0.120.9 34.7

44.3 0.122.1 31.7

46.1

1.820.8 27.8

49.5 1.421.3 29.1

48.2

CBA ECs + IFNγCBA ECs

CFSE

CFSE-labelled C57BL/6 DCs

MHC transfer between ECs and DCs is unidirectional

DCs acquire MHC class I and II molecules in vivo

(+PBS) (+IFNγ) (+IFNγ)

(+ PBS) (+IFNγ)immatur e DCs matur e DCs

(+IFNγ) (+PBS)

(+PBS)

78 .6

0.6 7.5

13 .3 92 .2

3.1 1.2

3.4 96 .7

2.8 0

0.5 99 .0

0.6 0

0.4

97 .9

2.0 0

0.196 .2

3.8 0

0.191 .5

3.5 2.0

3.076 .0

2.1 12.0

9.9

CFSE

B H-2Ab CFSE + H-2AbCFSE

a

PBS + immatureDCs-CFSE

b c

d

IFNγ + immatureDCs-CFSE

fe

h ig

+ PBS matureDCs-CFSE

d

g

IFNγ + immatureDCs-CFSE

+ PBS matureDCs-CFSE

ih

fe

a

+ PBS immatureDCs-CFSE

C

cb

H-2Kb CFSE + H-2K bCFSE

CD4CD8

Immature recipient DC

Mature recipient DC presenting donor MHC directly and indirectly

Donor parenchymal cells

Linking direct and indirect pathway T cells

Help or suppression

Lymph Node

Conclusions

• Dendritic cells acquire MHC class I and class II molecules from other DCs and from ECs

• The acquired MHC molecules are recognised by T cells

• MHC acquisition by DCs occurs in vivo, as well as in vitro

• This raises the possibility that direct alloresponses are maintained after donor DCs

are deleted

• This phenomenon may provide a mechanism whereby T cells with indirect allospecificity

can help or suppress T cells with direct anti-donor allospecificity.

Speculation….. the acquisition of MHC:peptide complexes by DCs traffickingthrough tissues provides a failsafe mechanism to ensure the presentation in lymphoid tissue of the viral peptide:MHC complexes that are most highly represented in the infected tissue…

an alternative to cross-priming….?

CD4CD8

Immature recipient DC

Mature recipient DC presenting viral peptide:class I complexes to CD8+ T cells

infected parenchymal cells

DC MHC acquisition as an alternative to cross-priming

Help

Lymph Node