Transplantation Immunology Current Status Volker Daniel, MD Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg,

Transplantation Immunology Current Status

Volker Daniel, MDInstitute of Immunology, Department of Transplantation Immunology,

University of Heidelberg,Im Neuenheimer Feld 305

D-69120 HeidelbergGermany

[email protected]

Transplantation Immunology

• TPL (numbers, overview, history)• HLA• Preoperative tests (prophylaxis of rejection)• Mechanisms of rejection (solid organs)• Immunosuppression• Tolerance (induction)• Clinical complications (infection, cancer)• Bone marrow and stem cell transplantation• Xenotransplantation• Artifical organs


• TPL (general aspects, numbers, overview, history)• HLA• Preoperative tests (prophylaxis of rejection)• Mechanisms of rejection (solid organs)• Immunosuppression• Tolerance (induction)• Clinical complications (infection, cancer)• Bone marrow and stem cell transplantation• Xenotransplantation• Artifical organs

Organ FailureAlternative Treatment Strategies

Mechanical organ replacement• Dialysis, bioartificial liver, cardiovascular device

Artificial organs• Tissue engineering, therapeutical cloning

Organs of other species• Xenotransplantation

Benefit of Transplantation

• nearly unrestricted quality of life

• rehabilitated in profession and social relationships

Expenses

Dialysis Renal transplantation1st year 50,000 € 50,000 €2nd year 50,000 € 2,500 €3rd year 50,000 € 2,500 €4th year 50,000 € 2,500 €

Pioneers of Transplantation

• Joseph Murray (kidney, 1954)• James D. Hardy (lung, 1963)• Richard Lillehei (pancreas, 1966)• Christian Barnard (heart, 1967)• Thomas E. Starzl (liver, 1967)

• Jean Dausset (HLA, 1958)• Jon van Rood (Eurotransplant, 1967)

International Transplant Records

Longest surviving recipient with continuous function:

Kidney 39 yearsLiver 32 yearsBone Marrow 29 yearsHeart 24 yearsPancreas 21 yearsLung 18 yearsIntestine 13 years

Clinical Transplants 2001; 279-318

Postmortale Organspenden (Stand: 02/06)

www.dso.de

www.ctstransplant.org



Genetic organization of the MHC in humans and the mouse

HLA class II

Expression of MHC Antigens

MHC subregion H-2 HLA

Tissue distribution of antigen

K, D, L, A, B, C All nucleated cells and platelets, erythrocytes

(mouse)

I-A I-E

D B lymphocytes Macrophages Monocytes

Epithelial cells (?) Melanoma cells

Activated T cells (human)

Association between HLA and

susceptibility to

autoimmune disease

Population studies show association of susceptibility to insulin-dependent diabetes mellitus

(IDDM) with HLA genotype

Family studies show strong linkage of susceptibility to insulin-dependent diabetes mellitus (IDDM) with HLA genotype



Microlymphocytotoxicity Assay

Separation of lymphocytes using densitiy gradient centrifugation

Separation of lymphocytes in T and B cells using Dynabeads

Dotting lymphocytes on Terasaki microtrays predotted with HLA antisera, Incubation period of 30 min

Addition of rabbit serum (complement), Incubation period of 60 min

Addition of acridinorange and ethidiumbromide, Incubation period of 15 min

Fluorescence microscopy

Mikrolymphocytotoxicity test

Negative Positive

Molecular-Based Techniques for HLA Typing

• RFLP (Restriction Fragment Length Polymorphism)

• PCR-SSO (PCR - Sequence Specific Oligonucleotide Hybridization)- Dot Blot (Standard Procedure) = amplified DNA is dotted- RDB (Reverse Dot Blot = oligos are dotted)

• PCR-SSP (ARMS) (PCR - Sequence Specific Primers)

• SBT (Sequence Based Typing)

• PCR-RFLP u.a.

HLA-Typing; Degrees of Resolution

Resolution Digits Indication Method

Low 2 kidney Tx, platelettransfusion

serology, PCR-SSO, PCR-SSP,

RFLP

High 4 Bone marrow Tx,forensics, disease

associationstudies

PCR-SSO, PCR-SSP, SBT

Nomenclature of HLA alleles Nomenclature Indicates

HLA the HLA region and prefix for an HLA gene

HLA-DRB1 a particular HLA locus i.e. DRB1

HLA-DRB1*13 a group of alleles which encode the DR13 antigen

HLA-DRB1*1301 a specific HLA allele

HLA-DRB1*1301N a null allele

HLA-DRB1*130102 an allele which differs by a synonymous mutation

HLA-DRB1*13010102

an allele which contains a mutation outside the coding region

HLA-DRB1*13010102N

a null allele which contains a mutation outside the coding region

Polymorphism of HLA June 2006

http://www.ebi.ac.uk/imgt/hla/stats.html

HLA class I

Gene A B

Allele 451 782

Protein 358 672

Nulls 36 25

HLA class II

Gene DRB1 DQB1

Allele 522 71

Protein 430 55

Nulls 7 1

HLA-A HLA-B HLA-DRB11 7 12 8 33 13 411 14 723 15 824 18 925 27 1026 35 1129 37 1230 38 1331 39 1432 40 1533 41 1634 4236 4443 4566 4668 4769 4874 4980 50

515253545556575859677378818283

http://www.anthonynolan.com/HIG/nomenc.html

Full List of HLA-A, -B, and -DR alleles (2 digits) assigned as of April 2003

HLA-B B*0808N B*1508 B*1548 B*1812 B*3506 B*3545 B*3922 B*4032 B*4415 B*4807 B*5129 B*5604

B*070201 B*0809 B*1509 B*1549 B*1813 B*3507 B*3701 B*3923 B*4033 B*4416 B*4901 B*5130 B*560501

B*070202 B*0810 B*1510 B*1550 B*1814 B*3508 B*3702 B*3924 B*4034 B*4417 B*4902 B*5131 B*560502

B*070203 B*0811 B*151101 B*1551 B*1815 B*350901 B*3703N B*3925N B*4035 B*4418 B*4903 B*5132 B*5606

B*0703 B*0812 B*151102 B*1552 B*1817N B*350902 B*3704 B*3926 B*4036 B*4419N B*5001 B*5133 B*5607

B*0704 B*0813 B*1512 B*1553 B*1818 B*3510 B*3705 B*3927 B*4037 B*4420 B*5002 B*5134 B*5608

B*0705 B*0814 B*1513 B*1554 B*2701 B*3511 B*3801 B*400101 B*4038 B*4421 B*5004 B*520101 B*5609

B*0706 B*0815 B*1514 B*1555 B*2702 B*3512 B*380201 B*400102 B*4039 B*4422 B*510101 B*520102 B*5610

B*0707 B*0816 B*1515 B*1556 B*2703 B*3513 B*380202 B*400103 B*4040 B*4423N B*510102 B*520103 B*5611

B*0708 B*0817 B*1516 B*1557 B*2704 B*3514 B*3803 B*4002 B*4042 B*4424 B*510103 B*520104 B*570101

B*0709 B*0818 B*15170101 B*1558 B*270502 B*3515 B*3804 B*4003 B*4043 B*4425 B*510104 B*5202 B*570102

B*0710 B*0819N B*15170102 B*1560 B*270503 B*3516 B*3805 B*4004 B*4044 B*4426 B*510105 B*5203 B*5702

B*0711 B*1301 B*1518 B*1561 B*270504 B*3517 B*3806 B*4005 B*4045 B*4427 B*510201 B*5204 B*570301

B*0712 B*1302 B*1519 B*1562 B*270505 B*3518 B*3807 B*40060101 B*4101 B*4428 B*510202 B*5205 B*570302

B*0713 B*1303 B*1520 B*1563 B*270506 B*3519 B*3808 B*40060102 B*4102 B*4429 B*5103 B*5301 B*5704

B*0714 B*1304 B*1521 B*1564 B*2706 B*3520 B*3809 B*4007 B*4103 B*4430 B*5104 B*5302 B*5705

B*0715 B*1306 B*1523 B*1565 B*2707 B*3521 B*390101 B*4008 B*4104 B*4431 B*5105 B*5303 B*5706

B*0716 B*1307N B*1524 B*1566 B*2708 B*3522 B*390103 B*4009 B*4105 B*4432 B*5106 B*5304 B*5707

B*0717 B*1308 B*1525 B*1567 B*2709 B*3523 B*390104 B*4010 B*4106 B*4433 B*5107 B*5305 B*5708

B*0718 B*1309 B*1526N B*1568 B*2710 B*3524 B*390201 B*4011 B*4201 B*4434 B*5108 B*5306 B*5709

B*0719 B*1310 B*1527 B*1569 B*2711 B*3525 B*390202 B*4012 B*4202 B*4435 B*5109 B*5307 B*5801

B*0720 B*1311 B*1528 B*1570 B*2712 B*3526 B*3903 B*4013 B*4204 B*4501 B*5110 B*5308 B*5802

B*0721 B*1401 B*1529 B*1571 B*2713 B*3527 B*3904 B*401401 B*4205 B*4502 B*5111N B*5309 B*5804

B*0722 B*1402 B*1530 B*1572 B*2714 B*3528 B*3905 B*401402 B*44020101 B*4503 B*5112 B*5401 B*5805

B*0723 B*1403 B*1531 B*1573 B*2715 B*3529 B*390601 B*4015 B*44020102S B*4504 B*511301 B*5402 B*5806

B*0724 B*1404 B*1532 B*1574 B*2716 B*3530 B*390602 B*4016 B*440202 B*4505 B*511302 B*5501 B*5807

B*0725 B*1405 B*1533 B*1575 B*2717 B*3531 B*3907 B*4018 B*440203 B*4506 B*5114 B*5502 B*5808

B*0726 B*140601 B*1534 B*1576 B*2718 B*3532 B*3908 B*4019 B*440301 B*4601 B*5115 B*5503 B*5901

B*0727 B*140602 B*1535 B*180101 B*2719 B*3533 B*3909 B*4020 B*440302 B*4602 B*5116 B*5504 B*670101

B*0728 B*15010101 B*1536 B*180102 B*2720 B*3534 B*3910 B*4021 B*4404 B*47010101 B*5117 B*5505 B*670102

B*0729 B*15010102N B*1537 B*1802 B*2721 B*3535 B*3911 B*4022N B*4405 B*47010102 B*5118 B*5507 B*6702

B*0730 B*150102 B*1538 B*1803 B*2723 B*3536 B*3912 B*4023 B*4406 B*4702 B*5119 B*5508 B*7301

B*0731 B*150103 B*1539 B*1804 B*2724 B*3537 B*3913 B*4024 B*4407 B*4703 B*5120 B*5509 B*7801

B*0801 B*150104 B*1540 B*1805 B*2725 B*3538 B*3914 B*4025 B*4408 B*4704 B*5121 B*5510 B*780201

B*0802 B*1502 B*1542 B*1806 B*350101 B*3539 B*3915 B*4026 B*4409 B*4801 B*5122 B*5511 B*780202

B*0803 B*1503 B*1543 B*1807 B*350102 B*3540N B*3916 B*4027 B*4410 B*4802 B*5123 B*5512 B*7803

B*0804 B*1504 B*1544 B*1808 B*3502 B*3541 B*3917 B*4028 B*4411 B*4803 B*5124 B*5513 B*7804

B*0805 B*1505 B*1545 B*1809 B*3503 B*3542 B*3918 B*4029 B*4412 B*4804 B*5126 B*5601 B*7805

B*0806 B*1506 B*1546 B*1810 B*3504 B*3543 B*3919 B*4030 B*4413 B*4805 B*5127N B*5602 B*8101

B*0807 B*1507 B*1547 B*1811 B*3505 B*3544 B*3920 B*4031 B*4414 B*4806 B*5128 B*5603 B*8201

B*8202

B*8301

Full List of HLA-B alleles assigned as of April 2003

New HLA Antigens und Alleles

http://www.ebi.ac.uk/imgt/hla/intro.html

Meyer M, Czachurski D, Tran TH, Opelz G, Mytilineos J.

A new PCR-SSP typing method for six single-nucleotide polymorphisms impairing the blood-clotting cascade as well as T-cell stimulation.Tissue Antigens. 2005 Dec;66(6):650-5.

Czachurski D, Scollo A, Skambraks A, Perichon AM, Scherer S, Tran TH, Opelz G, Grappiolo I, Mytilineos J.

Description and characterization of two new HLA alleles, B*4051 and DRB1*1364, identified by sequence-based typing.Tissue Antigens. 2005 Aug;66(2):151-5.

Czachurski D, Scherer S, Gehrke S, Laux G, Opelz G, Mytilineos J.

Identification of two new HLA alleles: B*3546* and B*5611*. How reliable are the published HLA-B intron 2 sequences?Tissue Antigens. 2004 Oct;64(4):500-5.

Czachurski D, Opelz G, Mytilineos J.

A new HLA-DRB allele (DRB1*15014) identified in a Caucasian individual.Hum Immunol. 2003 Feb;64(2):310-3.

Czachurski D, Rausch M, Scherer S, Opelz G, Mytilineos J.

Characterization of a new HLA-A allele, A*0256, identified in a Caucasian individual.Tissue Antigens. 2002 Aug;60(2):180-3.

Lancet. 2005 Apr 30-May 6;365(9470):1570-6.

Non-HLA transplantation immunity revealed by lymphocytotoxic antibodies.

Opelz G; Collaborative Transplant Study.

Figure 2. 10-year follow-up of kidney grafts from HLA-identical sibling donors

Transplantation. 2002 Apr 27;73(8):1269-73.

Kidney graft failure and presensitization against HLA class I and class II antigens.

Susal C, Opelz G.

Figure 1. Influence of ELISA-detected pretransplant IgG-anti-HLA class I and class II antibodies on cadaver kidney graft survival. Recipients possessing both anti-HLA class I and class II antibodies (I+/II+) had a significantly lower graft survival rate than antibody-negative recipients (I-/II-) (log-rank P <0.0001). Anti-HLA class I-positive/class II-negative (I+/II-) and anti-HLA class I-negative/class II-positive recipients (I-/II+) showed surprisingly good graft success rates.

N Engl J Med. 1994 Mar 24;330(12):816-9.

The influence of HLA compatibility on graft survival after heart transplantation. The Collaborative Transplant Study.

Opelz G, Wujciak T.

Figure 1. Actuarial Survival Rates of First Heart Transplants According to the Number of HLA-A, B, or DR Mismatches. The numbers of mismatched antigens and the numbers of grafts studied are indicated for each curve.

The Eurotransplant International Foundation is responsible for the

mediation and allocation of organ donation procedures in

Austria Belgium Germany Luxemburg The Netherlands Slovenia

In this international collaborative framework, the participants include all transplant hospitals, tissue-typing laboratories and hospitals where organ donations take place. The Eurotransplant region numbers well over 118 million inhabitants.

Eurotransplant

Transplantation Centers

Dialysis Patients on Waiting List

• ABO blood group

• HLA-A, -B, -C, -DR, -DQ alleles

• Panel reactive antibodies

Dialysis Patients on Waiting List

Every 3 months serum screening for HLA antibodies

Results to Eurotransplant

Sera of immunized patients shipped to transplantation centers for future crossmatches

Sensitization Against HLA Antigens

• Previous Transplantation

• Pregnancy

• Blood Transfusions

• Virus Infections

Sensitization of Dialysis Patients

Sensitization Number of Patients (n=358)• 0% 282• 1-10% 33• 11-20% 13• 21-30% 6• 31-40% 5• 41-50% 7• 51-60% 4• 61-70% 2• 71-80% 2• 81-90% 2• 91-100% 1

Retransplants (Kidney)

Previous Transplants Number of Patients (n=358)

0 293

1 49

2 16

3 0

Cross Talk

Eurotransplant

Donor Center Recipient Center

HLA Typing Lab

Kidney DonorABO blood group and HLA typing


Crossmatch list from ET with prospective recipients

Crossmatches

Results to ET

Patient with negative crossmatch and highest score receives kidney

Criteria for Kidney Allocation

• ABO blood group• Compatibility in HLA-A, -B, and -DR alleles• HLA mismatch probability• Waiting time• Preservation time• National organ exchange balance

Priority

• Children (<16 years of age)

• Patients immunized against HLA antigens

• High urgency patients

• Patients with combined transplantations

Kidney DonorABO blood group and HLA typing


Crossmatch list from ET with prospective recipients

Crossmatches

Results to ET

Patient with negative crossmatch and highest score receives kidney

Crossmatch and Transplantation

Crossmatch Result T B U U + DTT

#Tx, PRA Transplantation

- - - - y+ - + + n+ - + - y+ + + + n- + (+) (+) 1st Tx, <50% PRA y- + (+) (+) >1st Tx, >50% PRA n

T = T lymphocytes; B = B lymphocytes, U = unseparated lymphocytes; DTT = Dithiotreitol#Tx = number of transplantationPRA = Panel reactive antibodiesy = yes; n = no+ = positive; - = negative; (+) = weakly positive

Preservation Time

• Kidney 24 - 72 hours

• Heart 4 - 8 hours

• Liver - 24 hours

• Pancreas - 12 hours

• Lung - 6 hours

Definition of Immunogenetic Relationsship (Transplant - Host)Term Origin of Graft Fate of Graft

Autograft Donor = recipient Transplant is not rejected

Isograft Genetically indenticalindividuals (twins)

Transplant is not rejected

Allograft Genetically differentindividuals of the samespecies

Transplant is rejected after 9-14 days(first set) of accelerated whenretransplant (second set)

Xenograft Different species Transplant is rejected rapidly

Methods of Transplantation

• Cellular transplant: injection of cell suspension

• free transplant: no vascular anastomosis

• stemmed transplant: temporary vascular stem, removed after take of the graft

• organ transplantation: vascular anastomosis

Location of the Transplant

• orthotopic: at the original location

• heterotopic: at another location



IL-1

Ag

DC1

IL-1

TH1

TH2

TH2

TH2

B

TH1

TH1

TH1

TH2

TzTz

TzTz

K/NK

Pl

Pl

Pl

AkTs

+ -

IL-2

IL-2

IL-2, IL-12, IFN-y

IL-2, IL-12, IFN-y

Perforin

Granzyme AZz+

IL-4

IL-5

IL-6

IL-10

IL-13

K/NK

K/NKK/NK

Perforin

Granzyme A

Zz+

Tm

Bm

Nekrose

Apoptose

Neopterin

Nekrose

Apoptose

DC2

KomplementlyseOpsonisierung und ADCCPhagozytose

Kinetic of Immune Response

Immunosuppressive Drugs

• Antibodies against lymphocytes(Anti-CD3, ATG, Anti-CD25)

• Calcineurin inhibitors(Cyclosporine, Tacrolimus [=FK-506])

• Corticosteroids

• Antimetabolites (cytotoxic)(Azathioprine, Mycophenolate Mofetil)

Anti-CD3, Anti-CD4, Anti-CD25

• heterologous monoclonal antibodies against lymphocytes

• source: mouse• Induction of ADCC, apoptosis and blockade of cell

function• bridging of activated cytotoxic T cells and T helper

cells killing

• Induction of T suppressor cells

ATG Treatment During Heart Transplantation

0

50

100

150

200

250

300

350

-0,5 0 0,5 4 12 24 72 96

Hours before, during and after transplantation

cells

/µl

CD3 (T cellc)

CD16 (NK cells)

CD19 (B cells)

Recombinant IL-2R Antibody

Anti-IL2R Treatment of Renal Transplant Recipients

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

18,00

0 7 14 21 28 42 57 72 82 103

Days pre and post transplantation

% C

D3+

CD

25+

T ly

mph

ocyt

es

CD25*A-Region

CD25*B-Region

Corticosteroids

• Inhibit IL-1ß and IL-2 synthesis

• Redistribution of CD4+ lymphocytes from the circulation to other compartments

Mycophenolate Mofetil

• Inhibits T and B cell proliferation by blocking the production of Guanosin nucleotids for DNA synthesis

• Inhibits Inosin-Monophosphat-Dehydrogenase (IMP-DH) and thereby production of Guanosin nucleotids for DNA synthesis

• Specific for IMP-DH-Isoforms in T and B lymphocytes

• Inhibits glycosylation of adhesion molecules ( Attachment of lymphocytes on endothelium, and invasion of leucocytes in allograft)

Cyclosporine

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

1 10 100 1000 10000

Days posttransplant

Cy

clo

sp

ori

ne

(m

g/k

g/d

ay

)

Daniel et al., Transplantation 2005, 79:1498

Methylprednisolone

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

1 10 100 1000 10000

Days posttransplant

Met

hyl

pre

dn

iso

lon

e (m

g/k

g/d

ay)


Mycophenolate mofetil

0

5

10

15

20

25

30

35

40

1 10 100 1000 10000

Days posttransplant

Myc

op

hen

ola

te m

ofe

til (

mg

/kg

/day

)


Mycophenolate mofetil

0

5

10

15

20

25

30

35

40

1 10 100 1000 10000

Days posttransplant

Myc

op

hen

ola

te m

ofe

til (

mg

/kg

/day

)


Plasma IL-2

0

5

10

15

20

25

30

35

1 10 100 1000 10000

Days posttransplant

Pla

sm

a I

L-2

(p

g/m

l)


Plasma IL-10

0

2

4

6

8

10

12

14

16

1 10 100 1000 10000

Days posttransplant

Pla

sma

IL-1

0 (p

g/m

l)


Steroids

X

IL-1X

IL-1X

XX

Ag

DC1

TH1

TH2

TH2

TH2

B

TH1

TH1

TH1

TH2

TzTz

TzTz

K/NK

Pl

Pl

XAkTs

+ -

IL-2

IL-2

IL2, IL-12, IFN-y

IL-2, IL-12, IFN-y

Perforin

Granzyme AZz+

IL-4

IL-5

IL-6

IL-10

IL-13

K/NK

K/NKK/NK

Perforin

Granzyme A

Zz+

Tm

Bm

Necrosis

Apoptosis

Complement lysis

Opsonization and ADCC

Phagocytosis

Ciclosporine

Steroids

Mycophenolate

Mycophenolate

X

X

X

X

X

XAnti-CD3 mAb

Anti-CD3 mAb

XX

XX

X

Nekrosis

ApoptosisXXX

Mycophenolate

Mycophenolate

XXX

Pl

X

XAnti-CD3 mAb

Steroids

XX

DC2

Steroids

X

http://www.thedrugmonitor.com/transplantpharmacy.html



Rejection of Allografts

• hyperacute: within minutes

• acute: 5-9 days posttransplant

• chronic: lingering for weeks and months

Vascular Rejection

Interstitial Rejection

Chronic Rejection



Undersuppression

• acute rejection

Oversuppression

• drug toxicity

• infections

• cancer

Cause of DeathFirst Cadaver Kidney Transplant

First Year(n=832)

2 – 5 Years(n=1037)

5-10 Years(n=1063)

Infection 38.1% 16.1% 12.6%Cardiac 22.8% 27.6% 30.5%CVA 6.9% 8.6% 10.9%Cancer 4.5% 16.8% 17.0%

CTS-Study

Posttransplantation Lymphoproliferative Disease (PTLD)

• EBV-induced B cell lymphoma; loss of antiviral control due to strong postoperative immunsuppression

• Reduction or withdrawl of immunsuppressive drugs

Incidence of B Cell Lymphoma

• Normal population: 10/100.000

• During 1. year after kidney transplantation: 250/100.000

• During 1. year after heart transplantation: 1.200/100.000

Am J Transplant. 2004 Feb;4(2):222-30.

Lymphomas after solid organ transplantation: a collaborative transplant study report.

Opelz G, Dohler B.

Figure 1: Ten-year incidence of non-Hodgkin lymphomas (NHLs) in cadaver kidney recipients. Transplants were performed from 1985 to 2001. Expected incidence was based on a nontransplant population of same age and sex distribution and same geographical origin. Relative risk (RR) during the first post-transplant year was 24.6 and yearly RR ranged from 7.3 to 11.2 during the following 9 years.

Lymphocyte Subpopulation Monitoring

• CD3 T lymphocytes

• CD3/25 activated IL-2R+ T lymphocytes

• CD3/DR activated HLA-DR+ T lymphocytes

• CD4 T helper lymphocytes

• CD4/DR activated HLA-DR+ T helper lymphocytes

• CD8 suppressor/cytotoxic T lymphocytes

• CD8/DR activated HLA-DR+ T lymphocytes

• CD16 natural killer cells

• CD19 B lymphocytes

• DR HLA-DR+ monocytes, B lymphocytes, activated Tlymphocytes

J Heart Lung Transplant. 2005 Jun;24(6):708-13.

Effectivity of a T-cell-adapted induction therapy with anti-thymocyte globulin (sangstat).

Koch A, Daniel V, Dengler TJ, Schnabel PA, Hagl S, Sack FU.

Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany; Department of Cardiology, University of Heidelberg, Heidelberg, Germany.

BACKGROUND: Cytolytic induction therapy with anti-thymocyte globulin (ATG) should induce effective immunosuppression, with a low rate of rejection in the initial phase after heart transplantation. Induction therapy with ATG allows post-operative renal recovery without the negative effects of highly nephrotoxic cyclosporine levels. An increased rate of infection is a common problem, however, and has been associated with "over-immunosuppression" early after transplantation. Therefore, we investigated whether reduced T-cell-adapted ATG induction therapy could be performed without increasing the risk of graft loss by rejection and whether reductions in infection rates and costs are possible. METHODS: Between March 1999 and December 2002, T-cell-adapted ATG induction therapy with ATG (Sangstat) (1.5 mg/kg) was given to 62 heart transplant recipients (study group) starting on post-operative Days 1 to 6. T-lymphocyte sub-populations were screened daily using flow cytometry. If total lymphocytes were <100/mul (reference 1,300 to 2,300/mul), T-helper lymphocytes (CD4(+)) <50/mul (reference >500/mul) and T-suppressor cells (CD8(+)) <50/mul (reference >300/mul), then no ATG was given. Further immunosuppression was continued with triple therapy consisting of methylprednisolone, azathioprine and cyclosporine. An historic group of heart transplant recipients given a full-dose ATG regimen for 8 days served as controls. These recipients were treated with ATG (Merieux 1.5 mg/kg) until reaching monoclonal cyclosporine levels of >300 mg/dl. Additional immunosuppressive treatment did not differ. Patients in both groups received systemic antibiotics (Imipenem) peri-operatively. Results of routine endomyocardial biopsies and rates of infections were examined. RESULTS: Study group patients were older (52 +/- 10 vs 49 +/- 14 years). In the study group, mean cumulative ATG dose was reduced significantly to 596 +/- 220 mg (p < 0.05) for 3.9 +/- 1.6 days compared with 1,159 +/- 376 mg for 6.9 +/- 1.1 days in the control group. The rate of cytomegalovirus (CMV) seroconversion was 23% in the study group compared with 13% in the control group. Rate of deep sternal infections was lower in the study group (1.6% vs 3.2%). The mean rejection rate in the first 3 months was 0.4 +/- 0.7 for the study patients (185 biopsies) vs 1.1 +/- 1.7 for controls (237 biopsies). All biopsies with ISHLT Grade >2 were treated successfully with 1,000 mg of methylprednisolone intravenously for 3 days. Both groups showed a similar 1-year survival rate (study 88%, control 89%). CONCLUSIONS: T-cell-adapted ATG induction therapy can be a helpful tool for individualized immunosuppression. It is not associated with an increased rate of rejection. Lower doses of immunosuppression help to minimize the rates of infection. In addition, cytolytic induction therapy combined with reduced ATG results in significant cost reduction.



Induction of Tolerance

• Clonal deletion• T suppressor lymphocytes• Regulatory T lymphocytes• Antiidiotypic antibodies• Anergy• DC1/DC2 • Microchimerism• Indoleamine 2,3-dioxygenase

IL-1

Ag

DC1

IL-1

TH1

TH2

TH2

TH2

B

TH1

TH1

TH1

TH2

TzTz

TzTz

K/NK

Pl

Pl

Pl

AkTs

+ -

IL-2

IL-2

IL-2, IL-12, IFN-y

IL-2, IL-12, IFN-y

Perforin

Granzyme AZz+

IL-4

IL-5

IL-6

IL-10

IL-13

K/NK

K/NKK/NK

Perforin

Granzyme A

Zz+

Tm

Bm

Nekrose

Apoptose

Neopterin

Nekrose

Apoptose

DC2

KomplementlyseOpsonisierung und ADCCPhagozytose

Mechanism of Self-Tolerance for T Cells

Central (thymic)

• lack of positive selection: before CD4-, CD8-

• clonal deletion: at CD4+, CD8+

• central clonal anergy: at CD4+ or CD8+

Mechanisms of Self-Tolerance for T CellsPeripheral

clonal deletion: irregular activation

• crosslinking CD3/TCR with no APC

• crosslink CD3/TCR and class I together

• independent crosslinking of CD3/TCR and CD4

clonal anergy: incompatible activation

• IL-2 gene silenced

• IL-2 unresponsiveness

• low TCR/CD3

• low CD4/CD8

immunosuppression: complete activation in the presence of negative regulators

• nonspecific factors: TGF-ß, Th1-Th2, steroids

• cognate interactions: idiotypes

compartmentalization

Am J Transplant. 2005 Apr;5(4 Pt 1):746-56

Evaluation of T-cell receptor repertoires in patients with long-term renal allograft survival.

Alvarez CM, Opelz G, Giraldo MC, Pelzl S, Renner F, Weimer R, Schmidt J, Arbelaez M, Garcia LF, Susal C.

The mechanisms underlying long-term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T-cell receptor (TCR) repertoires in circulating T cells of patients with long-term (> or = 9 years) renal allograft survival with (LTS-IS) and without immunosuppression (LTS-NoIS). T cells of LTS patients exhibited strongly altered TCR Vss usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS-NoIS and 12 of 16 LTS-IS patients demonstrated oligoclonality in at least three or more TCR V beta families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well-functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long-term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T-cell subpopulations, such as regulatory T cells.

Figure 3: Percentages of oligoclonality in LTS-IS, LTS-NoIS, WF3, UC1, AR, CAN7, CR13 and dialysis patients and healthy controls.*p < 0.05, **p < 0.01, ***p < 0.001. Median, interquartile range (boxes) and range (whiskers) are shown.

J Exp Med. 1973;137:1142-1162

Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.

Ralph M. Steinman and Zanvil A. Cohn.

During the course of observation on the cells of mouse spleen that adhere to glass and plastic surfaces, it was clear that this population was quite heterogeneous. In addition to mononuclear phagocytes, granulocytes, and lymphocytes, we noticed a large stellate cell with distinct properties from the former cell types. In this paper, we describe the morphology, quantitation, and tissue distribution of this novel cell as identified in vitro. In following papers, we will further characterize it with respect to its functional properties in vitro, as well as its localization and properties in situ.

A dendritic cell sensing a lymphocyteOlivier Schwartz, Virus and Immunity Group, Institut Pasteur, Paris, France

Nature Cell Biology 2004; 6(3):188

Mechanisms of graft rejection

Lechler et al. Nat Med. 2005 Jun;11(6):605-13.

Selected strategies for tolerance induction now in clinical trials.

Lechler et al. Nat Med. 2005 Jun;11(6):605-13.

Kapsenberg, M. L. Nat. Rev. Immunol. 2003; 3:984.

Finding/Hypothesis I

• Myeloid DC (DC1) primed T lymphocytes differentiate into effector CD4+ or cytotoxic CD8+ cells mediating allograft rejection (Zou

et al. J Immunol 2000; 165:4388)

• Plasmacytoid DC (DC2) primed T lymphocytes differentiate into regulatory T cells promoting allograft tolerance (Kuwana et al.

Eur J Immunol 2001; 31:2547)

Rissoan et al., Science 1999; 283:1183

Cella et al., Nat Immunol 2000; 1:305

Finding/Hypothesis II

Immature DCs induce tolerance whereas mature DCs induce immunity.

Steinman et al. J Exp Med 2000; 191:411Dhodapkar et al. J Exp Med 2001; 193:233

Finding/Hypothesis

Tolerance results from: – T cell deletion– T cell silencing – Generation of regulatory T cells

Roncarolo et al. J Exp Med 2001; 193:F5Jonuleit et al. J Exp Med 2000; 192:1213

Generation of tolerogenic/regulatory DC

• Specific culture conditions for propagation of homogenous populations of immature DCs.

• Pharmacological manipulation of DCs to stabilize their immature/tolerogenic phenotype.

• Genetic modification of DCs to impair their stimulating ability/enhance their tolerogenicity.

Raimondi/Thomson; Contrib Nephrol 2005; 146:105

Nat Rev Immunol. 2004 Jan;4(1):24-34.

Dendritic cells: emerging pharmacological targets of immunosuppressive drugs.

Hackstein H, Thomson AW.

Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University Giessen, Langhansstr. 7, D-35392 Giessen, Germany. [email protected]

Hackstein and Thomson. Nat. Rev. Immunol. 2004; 4:24.



Problems of Xenotransplantation

• Natural IgM antinbodies

• MHC incompatibility

• PERVs

• Transfer of virus to human

• Incompatibility in secrected hormones

• Cross-reactive antibodies against MHC



Problems of Artifical Organs

• Energy supply

• Blood coagulation

• Replacing only part of organ functions

• Immunization of patient

• Induction of inflammation



Transplantation of

Bone marrow

• (from hip, narcosis)

Blood stem cells

• (from blood, without narcosis, G-CSF)

Problems of Bone Marrow Transplantation

• HLA-identical donor

• Graft versus host disease

• Host versus graft disease (rejection)

• Infection

Stammzellen

• Adulte menschliche Stammzellen: Sie werden aus Organen gewonnen. Das Arbeiten mit Ihnen ist erlaubt, weil dafür keine Embryonen "verwendet" werden. Die Zellen sind jedoch nicht so wandlungsfähig wie die embryonalen Stammzellen.

• Stammzellen aus Embryonen (ES): Die Zellen können sich in verschiedene Gewebetypen verwandeln. Somit öffnen sich ganz neue Möglichkeiten beispielsweise für die Therapie von Diabetes Mellitus, Alzheimer-Krankheit oder Morbus Parkinson. ES-Zellgewinnung ist jedoch umstritten, weil sie nur aus Embryonen gewonnen werden können. In Deutschland wird zur Zeit aus ethisch-moralischen Gründen darüber diskutiert, ob die deutschen Wissenschaftler für Stammzell Forschung und Gentherapie ES-Zellen aus Israel beziehen dürfen.

Transplantation Immunology Current Status Volker Daniel, MD Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg,

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