Gregg A. Hadley, Ph.D. Division of Transplantation Department of Surgery

Gregg A. Hadley, Ph.D.Division of Transplantation

Department of SurgeryUniversity of Maryland Medical School

Effector cells and tubulitis in the renal allograft.

Adaptive Immunity to Renal Allografts

Host

RenalAllograft

Cytokines

MØ activation

CytokinesDC licensing

Cytokines

Cognate help

CD4+

MHC II

Gran/PrfFas/FasL

CytokinesChemokines

CD8+

MHC I

ActivationDifferentiation

Allograft destructionCD8

CD8 Effectors

AntibodyPlasmaCell

Destruction of graft endothelium

ActivationDifferentiation

+ Complement Cascade

B cell

Interaction of CD8 effectors with graft epithelial compartments

•Attack of the renal tubular epithelium by graft infiltrating CD8 effector populations is a cardinal feature of clinical renal allograft rejection. •Current knowledge of CD8 effector properties is derived from studies of leukocyte:leukocyte interactions

•Consequently, the mechanisms underlying T cell/epithelial interactions remain poorly defined

ResponderSpleen Cells

Allogeneic RECStimulators

Allogeneic SCStimulators

40:180:1 20:1 10:1 5:10

10

20

30

40

50

60

70

0

10

20

30

40

50

60

40:180:1 20:1 10:1 5:1

Pe

rce

nt

Ly

sis

E:T Ratio

RECLC

TARGETS

Generation of kidney-restricted CTL

CD103

Eve

nts

anti-REC

anti-SC

CD103 defines a major subset of CD8 effectors generated against renal

epithelial cells

CD103 Background

•Integrin family heterodimer (formerly E7)

•Ligand (E-cadherin) is uniquely expressed by cells comprising epithelial layers

•Expressed by >95% of intraepithelial lymphocytes but poorly expressed by peripheral T cells (Brenner et al., Kilshaw et al., Lefrancois et al. ...)

•Previous studies have focused on the potential role of CD103 as a homing receptor that targets IEL to the gut mucosa •Role of CD103 in adaptive immune responses remains unclear

•CD103 promotes lysis of REC targets by in vitro generated CTL populations: Hadley et al., J Immunol, 1997; Rostapshova et al., Eur. J. Immunol, 1998.

Is CD103 expression by CD8 effector populations relevant to clinical renal allograft rejection?

Quad % Gated UL 25.41 UR 36.79 LL 35.87 LR 1.93

#2

Quad % Gated UL 22.46 UR 23.92 LL 49.91 LR 3.72

#3

CD103

CD

8

Quad % Gated UL 20.48 UR 34.80 LL 43.64 LR 1.07

#1

FACS analyses of transplant nephrectomy specimens

Robertson et al., Transplantation 71: 306-13, 2001Wong et al., Transplantation 75: 505-14, 2003

Does a causal relationship exist between CD103+CD8+ effectors and destruction of graft epithelial compartments?

CD103-deficient mice

•Mice with targeted disruption of the CD103 gene (Itgae, chromosome 11) on the BALB/c background

•Comparison of allograft rejection by CD103-/- mice vs. wild type mice provides a definitive means of documenting the contribution of CD103 to the rejection process

1) compared to mouse vascularized organ transplants, islet transplants are relatively easy to perform and thus amenable to experimentation

2) islets are specialized epithelial cells known to express high levels of the CD103 ligand, E-cadherin, and should be susceptible to destruction by CD103+CD8+ effectors

3) rejection of islet allografts transplanted into streptozotocin treated (diabetic) mice can be objectively assessed by serial measurement of blood glucose levels

4) islets can be transplanted under the renal capsule which allows recovery and analysis of graft infiltrating lymphocytes

Pancreatic islet transplant model

CD103+CD8+effectors are present at the graft site

35.28 9.6351.99 3.10

CD103

CD

8

100 101 102 103 104

055

18.1+3.0% CD103+ (n=4)

CD103

Eve

nts

Gated CD8+ T cells

CD103 knockout hosts are deficient in the capacity to reject pancreatic islet allografts

Gra

ft s

urv

ival

(%

)

Time (Days)

0

20

40

60

80

100

0 20 40 60 80 100

CD103-/- (n=17)Wild type (n=13)

Time (days)0 5 10 15 20 25

0

100

200

300

400

500

600

wild typeCD8 cells

CD103 -/-CD8 cells

Wild typeNon-CD8control

Adoptive transfer of CD8+ cells into CD103-/- hosts with long-term islet allografts

Transferred CD8 cells:

Blo

od G

luco

se (

mg/

dL

)

Islet allograft in wildtypehost at time of rejection

Islet allograft in CD103-/-

host at day 14 post-Tx

Conclusion

CD103 is required for destruction of graft epithelial compartments by CD8 cells, and appears to critically function at the level of intragraft homing of CD8 effectors

Do CD8+CD103+ effectors play a significant role in rejection of vascularized renal allografts?

Classical acute rejection model

Time (Days)

0 2 4 6 8 100

2

4

6

8

10

Gated CD8+ GIL

BN (RT1n) LEW (RT1l)

Ser

um

cre

atin

ine

(mg/

dL

)

Delayed rejection model

BN (RT1n) LEW (RT1l)

CsA 5 mg/kg/day for 10 days

Gated CD8+ GIL

Time (Days)

Ser

um

cre

atin

ine

(mg/

dL

)

0 10 20 30 40 500

2

4

6

8

10

CsA

Two-color ImmunostainingRed = E-cadherinBrown = CD103

Antibody blocking studies

CsA

day12

OX62 3.5mg IP POD 8

day28KTX

Per

cent

tub

ule

s w

ith

in

filt

rati

ng C

D8

cell

s

IgG1 Control

0

20

40

60

80

100

OX-62

P<0.01n=4

Anti-CD103 monoclonal antibody, OX-62, blocks movement of CD8 cells into tubules

Conclusion

CD103 promotes entry and/or retention of CD8 effectors in the graft renal tubules, consistent with a key role for CD103+CD8+ effectors in development of CAN.

What controls CD103 expression bygraft infiltrating CD8 effectors?

CD103 (Log10 Fluorescence)

Eve

nts

no TGF (day 1)

+ TGF (day 1)

+ TGF (day 2)

TGF-beta controls CD103 expression by allospecific CD8 effectors

In vitro

Per

cen

t C

D10

3+

0

20

40

60

80

Wildtype(n = 3)

DNRII(n=4)

In vivo

Graft Epithelium

Working Model

TGF-

CD8 effectors

IFN-

“Non-specific”inflammatory cells

Graft Site

Acknowledgements

Hadley LabElena RostapshovaYe FengRongwen YuanRiham El-Asady

Transplant PathologyCinthia Drachenberg