Transplant tolerance: progress and challenges Giuseppe Remuzzi - - PowerPoint PPT Presentation

Transplant tolerance: progress and challenges

Giuseppe Remuzzi

Cagliari, 30 aprile 2011

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Transplant of kidneys from one dog to another Brigham Hospital, Boston

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• Nothing is more ridiculous than

saying that all this could have been done with computers or with tissue

• cultures. That

s like saying you can live without breathing or without having blood. It s absolutely the most anti-intellectual thinking that ever existed

Joseph Murray, Nobel Prize Lecture, 1990

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• Joe

s the only guy who ever won a Nobel Prize for pursuing a hobby

Francis Moore

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23 Dicembre 1954, ore: 8.00 Ospedale Peter Bent Brigham di Boston

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Edith Helm and her sister Wanda Foster

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Analysis of UNOS data on patients with functioning kidney 1 year post transplant

Hariharan et al., N Eng J Med, 2000 USRDS, Am J Kidney Dis, 2009

GRAFT SURVIVAL AFTER RENAL TRANSPLANTATION IN THE PERIOD 1989-2005

years of transplant Projected median half-life

(years) 89 91 93 95

Deceased donor

30 20 10

9

2005 13 97 99 2001 2003

10 20 No rejection Rejection*

* within 1 year post-Tx 1995

18 9

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10 10 Basiliximab

(chimeric monoclonal antibody against IL-2 R)

THE PROMISE OF NOVEL IMMUNOSUPPRESSIVE AGENTS

Daclizumab

(humanized monoclonal antibody against IL-2 R)

CAMPATH-1H

(humanized anti-CD52 antibody - T and B cells depletion)

Everolimus

(m-TOR T cell proliferation inhibitor)

Belatacept

(IgG/CTLA4 fusion protein selective blocker of T cell activation) Kidney Tx

(Nashan et al., Lancet)

1997 1998

Kidney Tx

(Vincenti et al., N Engl J Med)

Kidney Tx

(Calne et al., Lancet)

2003 2005

Heart Tx

(Eisen et al., N Engl J Med)

Kidney Tx

(Vincenti et al., N Engl J Med)

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11 11

20 40 60 100 80 Tx patients with tumor (%) 10 20 30 40 Survival post-Tx (years)

RISK OF DEVELOPING A TUMOR IN TRANSPLANT RECIPIENTS

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12 12

New drugs are not necessarily better drugs

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Remuzzi et al., J Am Soc Nephrol, 2007

13 15,12 euro Patient death

Months after transplantation

Death-censored graft loss

HR: 0.89 (95%CI 0.32 to 2.46), p=0.83

6 12 18 24 30 36 42 48 54 60 66 72

Azathioprine Mycophenolate mofetil

HR: 0.96 (95%CI 0.28 to 3.31), p=0.95

Cumulative probability

0.1 0.2 6 12 18 24 30 36 42 48 54 60 66 72

Cumulative probability

0.1 0.2

Mycophenolate mofetil Azathioprine

0,62 euro

Daily cost dose

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One of the major questions remaining in clinical transplantation is whether it will be possible to induce states of true tolerance with little or no long-term drug therapy ideally one would like to alter the host s initial contact with the graft to promote a state

• f donor-specific unresponsiveness

Carpenter, N Engl J Med, 1993

THE TARGET: TRANSPLANTATION TOLERANCE

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Transplants in Rats Made Without Usual Drugs

By LAWRENCE K. ALTMAN

Kidney graft survival > 70 days 15

16 16 i.t. injection

(leukocytes x 10 8)

Time to Tx

(days)

1.8 7.0 14 7 Kidney graft survival

(days)

> 350 > 100

Knechtle et al., Transplantation, 1997

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17 17 RARE EXAMPLES OF SPONTANEOUS CLINICAL TRANSPLANTATION TOLERANCE AFTER IMMUNOSUPPRESSANT DISCONTINUATION 12 of 42 longest surviving liver recipients Years post-Tx Years off immunosuppressive drugs 17 23 16 22 18 19 24 17 14 13 26 14 15 13 13 10 7 4 3 2 2 2 1 1

Patients who are immunosuppression-independent show donor cell chimerism, i.e. persisting donor hematopoietic cells migrated from the transplanted organ into the host peripheral blood and bone marrow

18 IN KIDNEY TRANSPLANT RECIPIENTS GIVEN DONOR BONE MARROW INFUSIONS LONG-TERM GRAFT SURVIVAL IS IMPROVED

• Immunosuppressive therapy: OKT3, Tacrolimus, MMF, steroid
• Donor BMC infusion at day 4 and/or day 11 post-Tx
• Follow-up: 2.9 to 6.3 years (mean: 4.7 years)

Ciancio et al., Transplantation, 2001 50 70 60 80 90 100

% graft survival

(death censored)

Months post-Tx

24 12 36 48 60 72

DBMC (n = 63) Control (n = 219)

p = 0.039

19 19

Lewis rat recipient BN bone marrow or leukocyte infusion

(70-200 x 10 6)

CsA

(10 mg/kg/day, i.m.)

BN kidney transplant

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Days after cell infusion

A CLINICALLY APPLICABLE ANIMAL MODEL TO DISSECT MECHANISMS OF ORGAN TRANSPLANTION TOLERANCE

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Noris et al., J Am Soc Nephrol, 2001

20 20 PBMC BM cells Lewis rat recipient Groups Donor cell infused

(x 10 6)

Kidney graft survival

(days)

CsA alone BN- PBMC + CsA BN- BM + CsA BN-PBMC + CsA / 70 70 200 7, 7, 8, 9, 10, 10 16,17,25, 27,30,33,33,58,>70, >70>70,>70,>70,>70,>70,>70,>70 6,8,10,14,17,17,17,17,20,>60, >60,>60,>60 >70,>70,>70,>70

Noris et al., J Am Soc Nephrol, 2001

and kidney allograft Brown Norway

21 Kidney graft + PBL or BM Noris et al., J Am Soc Nephrol, 2001

INTRATHYMIC MICROCHIMERISM DETERMINES THE OUTCOME OF TOLERAZING PROTOCOLS TARGETED AT SOLID ORGAN TRANSPLANTATION

Lewis rat Brown Norway

% rats with microchimerism

Tolerance

Thymus Liver Spleen Blood

100 80 60 40 20

% rats with microchimerism

Rejection

Thymus Liver Spleen Blood

100 80 60 40 20

MHCII+

22

Noris et al., J Am Soc Nephrol, 2001

Time post-transplant (days)

100 80 60 20 40 60 40 20

MHC II+ cells depletion

% graft survival

Controls

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Control Thymectomy * Graft survival (days) 19, 55, >70, >70, >70, >70 5, 8, 15, 17, 17

* on the day of the kidney transplant

Noris et al., J Am Soc Nephrol, 2001

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How near are we to reaching the point where transplant tolerance will become a clinical reality?

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CHIMERISM AND TOLERANCE AFTER RENAL AND BONE MARROW TRANSPLANTATION

5 subjects with ESRD: Alport s syndrome (2), MPGN, PKD (2) Bone marrow (day 0) and kidney from one-haplotype-mismatched, living-related donors

Kawai et al., N Engl J Med, 2008

Cyclophosphamide Thymic irradiation Anti-CD2 mAb CsA Rituximab 60 mg/kg 700 cGy 0.1 mg/kg 5 mg/kg i.v. 375 mg/m2 BSA Preparative regimen CsA Prednisone 8-12 mg/d orally 2 mg/kg Post-Tx immunosuppression Follow-up after discontinuation of immunosuppression: 5 - 7 years

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LONG-TERM FOLLOW-UP (September 2010)

Original disease

1 2 3 4 5

Kidney survival (years) Patient n° Discontinuation immunosuppressive therapy (years)

* Relapse of MPGN # In 2004 underwent a second kidney Tx (stable renal function on conventional immunosuppressive therapy since then

Alport s syndrome MPGN type 1 PKD Alport s syndrome PKD 6.6 6.1 NO 4.9 3.8 7.3 7.2 * 10 days # 5.5 4.6

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LONG-TERM FOLLOW-UP

1 2 3

4 5

Donor specific antibodies Patient n° T cell unresponsiveness Yes Yes N/A

Yes Yes

Discontinuation immunosuppressive therapy (years) 6.6 6.1 NO

4.9 3.8

No No Yes

early humoral rejection

Yes*

late persistent

Yes*

late persistent

Porcheray, Am J Transpl, 2009

* co-development of autoantibodies

28 28

Unusually high incidence of acute humoral rejection despite the combined conditioned regimen

• f

thymic irradiation, cyclophosphamide, anti- CD2 mAb, cyclosporine and rituximab Risk of infections and aplasia and ultimately death outweigh the potential benefit of tolerance

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,

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Clinical update: immunosuppression minimisation

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BASILIXIMAB COMBINED TO LOW-DOSE RATG IN HIGH RISK KIDNEY TRANSPLANT RECIPIENTS

1 episode of biopsy-proven acute rejection on low-dose RATG plus basiliximab, and 2 on standard-dose RATG Less side effects* with the combined low-dose RATG and basiliximab

Ruggenenti et al., C J Am Soc Nephrol, 2006 * fever, elukopenia, anemia, CMV reactivation

Days post Tx

Kidney Tx Basiliximab (20 mg)

1 2

Randomization

Standard RATG (2 mg/kg/day)

3 4 5 6 7

Days post Tx

1 2 3 4 5 6 7

Low-dose RATG (0.5 mg/kg/day) Basiliximab (20 mg) Maintenance therapy with CsA, ST, MMF

,

3 Time post Tx

Kidney Tx

MP MP MP d1 d2 Bas - L-RATG*

Low CsA + low dose AZA or low MMF 20 mg -

• No acute humoral rejection
• Acute cellular graft rejection: 20%

Low SRL 0.5 mg/kg/die

3-5 mg/kg/day 75 mg/day 750 mgx2/day

* for 6 days

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The first 4 renal transplant recipients randomized to low SRL/MMF maintenance immunosuppressive regimen developed acute allograft rejection

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33 Time post Tx

Kidney Tx

MP MP MP d1 d2

Bas - L-RATG Low CsA + low dose AZA or low MMF

20 mg 0.5 mg/kg 3-5 mg/kg/day 75 mg/day 750 mgx2/day

?

Low SRL

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TOLERANCE Regulatory cells Effector T cells

Salama, Remuzzi, Harmon et al., J Clin Invest, 2001

REJECTION Regulatory cells Effector T cells

35 35

LW rat recipient BN rat donor Kidney Tx

Blood donor leukocytes

• 10

CsA

Collection of thymus

5

Isolation of graft infiltrating lymphocytes

30 60

Isolation of lymph nodes

days

DONOR-SPECIFIC T REGULATORY CELLS PLAY A ROLE IN TOLERANCE INDUCED BY PBMC INFUSION IN RATS

Graft survival prolongation: 47 % Tolerance: 53 %

• f total transplanted rats

Cavinato et al., Kidney Int, 2007

36 36

Cavinato et al., Kidney Int, 2007 BN rat donor

• 10

CsA

5 30 60 days

INDUCTION EARLY POST-Tx MAINTENANCE A A A

R A R N

?

graft

R R R

CD4+CD25+

Foxp3+

37 37

Cavinato et al., Transplantation, 2005 LW rat recipient BN rat donor Splenocytes Tolerant LW rat (>60 d post-)Tx Kidney

ADOPTIVE TRANSFER OF CELLS WITH REGULATORY ACTIVITY PROLONGS KIDNEY GRAFT SURVIVAL

Graft survival prolongation: 66, 83, > 100 days

HIGH CIRCULATING T REG COUNTS UNDER CAMPATH-1H DO NOT CONFER APPRECIABLE PROTECTION AGAINST GRAFT FUNCTION LOSS AND CHRONIC ALLOGRAFT REJECTION

Ruggenenti et al., Transplantation, 2008

• 3.5
• 3.0
• 2.5
• 2.0
• 1.5
• 1.0
• 0.5

∆GFR (ml/min/1.73m2/yr)

• 4.0
• 4.5

Treg+ Treg-

From months 6 to 30 post transplant

Treg+ Treg- CADI score*

(mean)

C4d staining* 5.2 4.3 1.0 0.4

*2 yrs post Tx

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Consequences of mTOR inhibition

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THE SPECIAL PROBLEM OF MEMORY

IFN-γ

Activated CD8+ memory Memory T cells contribute to allograft rejection through:

• Activation endothelial cells
• Help naive CD8, CD4 T cells and B cells

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41 A CRITICAL THRESHOLD OF DONOR-SPECIFIC MEMORY T CELLS IS REQUIRED TO RESIST TOLERANCE INDUCTION IN MICE

Adams et al., J Clin Invest, 2003

Donor skin graft + Tolerance protocol Donor skin graft + Tolerance protocol

Viral challenge single Viral challenge multiple

Jones et al., Transplantation, 2006 42

Calcineurin inhibitors Hydrocortisone Sirolimus, MMF, azathioprine IFN-γ blockade (in vitro) complete* partial negligible

42

When present at low level during T cell priming, CsA promotes the T cell commitment to a memory state

*

Valujskikh et al J Am Soc Nephrol, 2007

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44

Nauta et al, Blood, 2007

MSC

Expansion

T reg

Memory T cells Inhibition

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Nauta et al, Blood, 2007

MSC

NK cells

Cytotoxicity Proliferation

BCR

B cells Ig release Cell activation

T cells

Proliferation Cell activation Expansion Differentiation Maturation

DC T reg Inhibition Memory T cells

46 46

In a semi-allogeneic heart transplant pretransplant infusion of syngeneic MSC prolongs the survival of the graft through the expansion

• f

donor-specific CD4+CD25+Foxp3+ T-regulatory cells

Casiraghi et al., J Immunol, 2008

10 20 30 40 50 60 70 80 >100 days after transplant 20 40 60 80 100

no cell infusion MSC

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MSC INHIBIT ALLOANTIGEN-INDUCED IFN-γ IN HUMAN MEMORY T CELLS IN MLR

Casiraghi et al., 2010

IFNγ produciìng memory T cells

(spots/200 000 cells) 20 40 60 80 100 120 140 160 180 200

none MSC 1:2 MSC 1:10

* *

* p<0.05 vs none

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48

Timing of MSC infusion?

Timing of MSC infusion

Pre transplant Simulect (anti-CD25 antibody) could block MSC- induced Tregs which express high level of CD25 antigen Peri transplant Administration of MSC during induction therapy could result in RATG-mediated MSC lysis/apoptosis

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7 days post transplant

Timing of MSC infusion

RATG is present in patients serum at low dose and did not impair MSC availability and immunosuppressive properties This time coincides with the start homeostatic proliferation induced by lymphopenia

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MSC, given 7 day after transplantation, could limit lymphopenia-induced memory T cell proliferation while promoting the emergence of regulatory T cells Regulatory T cells Memory T cells Mesenchymal stem cells

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52 PBMC proliferation

(cpm x 10-3)

CsA* ng/mL 10 100 MPA* ng/mL 500 100 MP 10-6M none 300 350 + MSC 1:2 10 20 30 40 50

Perico, Casiraghi et al., Clin J Am Soc Nephrol, 2010

none

MSC IMMUNOMODULATORY FUNCTION IS NOT IMPAIRED BY IMMUNOSUPPRESSIVE DRUGS

52

* trough blood levels

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MSC TO PROMOTE RENAL TRANSPLANT TOLERANCE

A pilot explorative study (start with 3 patients)

Time post Tx (days) Kidney Tx

MP

1 2 Low CsA + Low MMF 4 3 5 6 7 Recipient MSC

(2 x 106/Kg i.v) L-RATG (0.5 mg/kg) Basiliximab (20 mg)

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LIVING-RELATED KIDNEY TRANSPLANTS COMBINED WITH RECIPIENT MSC INFUSION

• Male, 22 years old
• ESRD: unknown etiology (proteinuric nephropathy)
• RRT: hemodialysis (July 2007)
• ABO compatible (Rec: A, Rh+ Donor: 0, Rh+)
• 2 HLA mismatches ( HLA- A24, B35)
• Negative cross-match (FCXM)
• Negative anti-donor Abs (Bead Array Technique

Luminex)

Patient #1: D.D

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• Male, 34 years old
• ESRD: IgA nephropathy
• Pre-emptive transplant
• ABO compatible (Rec: A, Rh+ Donor: 0, Rh+)
• 2 HLA mismatches ( HLA- A30, B57)
• Negative cross-match (FCXM)
• Negative anti-donor Abs (Bead Array Technique

Luminex)

Patient #2: G.U.

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Treg/memory CD8+ T cells (ratio)

THE HIGH RATIO OF Treg/Tmemory COULD FAVOUR A STATE OF IMMUNE REGULATION

Patient #1 Patient #2 Recipients of a living-related donor kidney given Bas + Low RATG and maintenance therapy alone (N = 3). * P<0.05 vs pre-tx 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

days post transplant

Pre-tx 180 360

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Granzyme B

(spots/200.000 cells)

1 2 3 4 5 6 7 8 9 10 pre 180d 360d

*

Perico, Casiraghi et al., Clin J Am Soc Nephrol, 2010

Recipients of a living-related donor kidney given Bas + Low RATG and maintenance therapy alone (N = 3). *P<0.05 vs pre Patient #1 DD

MEMORY T CELLS*

Patient #2 GU

CYTOTOXICITY**

1 2 3 4 5 6

pre 180d 360d

% of lysis of donor cells 57

* anti-donor ** anti-donor CD8+ T cells

58

4 6 8 10 12 1 2 3 0 1 2 3 4 5 6 7 9 10 11 13 14 20 21 22 27 28 180 360 940

10.6 7.2 3.5 2.3 2.0 1.8 1.7 1.9 1.8 1.7 1.8 1.9 2.3 2.3 2.5 1.9 2.0

MSC infusion start MP pulses no biopsy (BT>12 min)

Serum creatinine (mg/dl)

Day post-Tx

Patient #1 D.D.

1.8 1.9

5 7 9 1 2 3 Day post-Tx

8.3

Serum creatinine (mg/dl)

0 1 2 3 4 5 6 7 9 10 11 13 14 15 16 17 18 19 20 21 27 180 360 575

5.3 2.7 1.7 1.5 1.49 1.43 1.66 1.60 1.791.87 1.85 2.01 2.34 2.1 1.87 1.58

Biopsy MSC infusion start MP pulses

Patient #2 G.U.

2.4

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2.2 1.8

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GRAFT INFILTRATING CELLS (day +15)

CD8+ T cells Patient #2: G.U. Controls Acute rejection (n=3) CD14+ monocytes CD4+ T cells (cells/field) 9.8 1.5 103 ± 35 39 ± 12 6.4 80 ± 35

Values are mean of 30 fields

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GRAFT INFILTRATING CELLS (day +15)

CD68+ macrophages Patient #2: G.U. Controls Acute rejection (n=3) Granulocytes CD20+ B cells (cells/field) 26.1 1.2 58 ± 2 5.5 ± 1.2 30.7 20 ± 16

Values are mean of 30 fields

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MSC INFILTRATE THE GRAFT (day +15)

Patient #2: G.U. Controls Acute rejection: 15 day post-tx Native control kidneys (n=4) CD105+ CD44+ cells

(cells/3 mm2)

1.5 ± 2.6

17

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Patient #1: DD, (1 yr post-Tx)

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GRAFT INFILTRATING CELLS (1 year)

CD8+ T cells

Patient #1: D:D. Controls Per-protocol biopsy (n=3)

CD14+ monocytes CD4+ T cells (cells/field) 5.5 6.5 14 ± 7 1.7 ± 1 1.6 9.5 ± 6

Values are mean of 30 fields.

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GRAFT INFILTRATING CELLS (1 year)

CD68+ macrophages

Patient #1: D.D. Controls Per-protocol biopsy (n=3)

Granulocytes CD20+ B cells (cells/field) 12.2 0.3 12 ± 5 5.8 ± 4.2 14.1 3 ± 2.4

Values are mean of 30 fields.

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65 Patient #1: D.D. Control Per-protocol biopsy: 2 years post-tx CD105+ CD44+ cells

(cells/3 mm2)

1.3 1.36

MSC INFILTRATE THE GRAFT (1 year)

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Farris et al., Personal Communication

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Patient #1 D.D.: Day 970 post- kidney transplant

Recipient in good health Stable graft function (s. creat 2 mg/dl) Current immunosuppressive therapy

CsA 75 mg + 75 mg /day MMF 750 mg b.i.d

• Patient #2 G.U.:

Day 600 post- kidney transplant

Recipient in good health

CsA 100 mg + 75 mg /day MMF 500 mg b.i.d methylprednisolone 8 mg/day

• Stable graft function (s. creat 1.8 mg/dl)

Current immunosuppressive therapy 68

69 Graft

MSC

Subclinical inflammatory environment Recruitment MSC activation

Release of proinflammatory /vasoactive mediators and complement activation Intragraft recruitment of PMNs Release of proinflammatory /vasoactive mediators

Vasoconstriction

• S. creatinine

Lymph node

IL-8 AA metabolites -TxA2 ? Complement

Engraftment syndrome reversible acute kidney injury in the second week after bone marrow or MSC and kidney transplantation

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71 B6 recipient mice autologous MSC infusion (500,000/mouse, i.v.) 1 2 3 4 5 6 7 ALS ALS + anti-CD25 MMF and CsA

Balb/c kidney

Transplanted groups: Untreated (n=10) MSC alone day +7 (n= 4) MSC+ ALS/MMF/CsA (n= 8)

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Balb/c kidney in B6 recipients

20 40 60 80 100

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 >100

130°

untreated MSC MSC+ ALS/MMF/Csa mice surviving (%) Time post Time post-

• transplant (days)

transplant (days)

MSC infusion

MSC do not affect kidney graft survival when given post transplant with or without immunosuppressive drugs 24 hours after infusion MSC were found in the transplanted kidneys

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Days post-transplant

10 20 30 40 50 60 70 80 90 100 5 10 15 20 25 30 35 40 45 50 55 60 >60

% survival

Untreated (n = 3) MSC at day -7 (n = 5) MSC at day -7 and day -1 (n = 5) MSC at day -1 (n = 2)

PRE-TRANSPLANT INFUSION OF MSC IN SENSITIZED MICE

B6 recipient mice Balb/c kidney Tx

Balb/c donor splenocyte (1,000,000/mouse i.v.) autologous MSC (500,000/mouse i.v.)

BUN (mg/dl)

20 40 60 80 100 120 140 160

7 10 20

days post-transplant

* day-7 and -1 * day-7 and -1 day-7 and -1 * day-1 day-1 day-1

untreated

GRAFT FUNCTION IN MICE GIVEN INFUSION OF AUTOLOGOUS MSC

* P<0.05 vs untreated

untreated

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EXPERIMENTAL DESIGN

Transplant

PKH26+ syngeneic MSC

• 3
• 2
• 1

1 2 3

PKH26+ syngeneic MSC

Group 1 (n=3) Group 2 (n=3)

B6 recipient mice Balb/c kidney Tx

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kidney spleen

Group 1 (MSC day -1) 208 ± 51 34 ± 8* 22 ± 9*

PKH 26+ MSC

Group 2 (MSC day +2) 5 ± 4 IN TRANSPLANTED MICE MSC ARE PREFERENTIALLY RECRUITED AT THE GRAFT SITE AND FEW MSC REACH LYMPHOID ORGANS (cells/mm2)

* P<0.05 vs group1

77

Tregs group #1 (MSC day -1) group #2 (MSC day +2)

2 4 6 8 10 12

% CD4+ Foxp3+ T cells

MSC GIVEN 2 DAYS AFTER TRANSPLANTATION FAILED TO EXPAND TREGS

°P<0.05 vs group 2

Splenocytes isolated from group #1 (MSC day -1) mice are able to prolong the survival of a Balb/c kidney transplant when transferred into naïve mice

°

PRE-TRANSPLANT MSC INFUSION IN KIDNEY TRANSPLANT RECIPIENTS

Time post Tx (days) Kidney Tx

MP

Low CsA + Low MMF Recipient MSC

(2 x 106/Kg i.v) L-RATG (0.5 mg/kg)

1 2 4 3 5 6 7

• 1

78

79 Patient #3 C.M.

4 6 8 10 12 1 2 3

0 1 2 3 4 5 6 7 9 10 90

10.3 4.0 1.8 1.3 1.3 1.1 1.2

MSC infusion Serum creatinine (mg/dl) Day post-Tx

• 1

1.2 1.2

120

1.3

150

1.2

80 80

Our clinical cell-based protocol in kidney transplant recipients was conceived as steps forward gain knowledge every 2 patients to look for the unexpected and ultimately identify a definite protocol that allows to create favorable conditions for tolerance, avoiding unwanted effects

81

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Immediately after Alonzo Mourning (famous basketball player from Miami) came on stage Black, about 7 feet tall, speaking like an old actor who everytime when he jokes is waiting for the reaction of the audience. Really funny, you can listen for hours to him Three years ago he received a kidney transplant

84

This year the Miami Heat won the NBA championship and they won for Alonzo Mourning

85

• Dr. Hardy said

I have done thousands of these, and I can do it with my eyes closed My response to him Well, make sure you at least keep one eye open when you are doing my surgery

Alonzo Mourning, World Transplant Congress, 2006

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For this study MSC are isolated and ex-vivo expanded according to Good Manufactoring Practice (GMP) by the Cell Therapy Laboratory G Lanzani , Hematology Unit, Ospedali Riuniti Bergamo It is authorized by the Italian Agency (AIFA)

• f the Ministry of Health to perform the

procedure (aM-189/2008)

88

MSC: ISOLATION AND EXPANSION

Nucleated cells from bone marrow aspirates are plated in the presence of 5% human platelet lysate Non-adherent cells are removed after 2-3 days After 13 days adherent cells are detached, replated, and cultured until confluence This procedure allows high rate of cell expansion in a single passage avoiding reagents of animal origin

Capelli et al., Bone Marrow Transplant, 2007 Capelli et al, Cytotherapy, 2009

89

MSC: IDENTIFICATION

Adherence to plastic in standard culture condition Phenotype by FACS analysis:

CD73, CD105, CD90 CD45, CD34, CD14 or CD11b, CD79a or CD19, HLA-DR Negative (< 2%): (International Society of Cell Therapy)

In vitro differentiation: osteoblasts, adipocytes, chondroblasts

Positive (> 95%):

Dominici et al., Cytotherapy, 2006

(by staining of in vitro cell culture)

90

MSC: CYTOGENETIC ANALYSIS

Normal karyotype found in 20 metaphases on 40 consecutive separate batches of MSC

1 batch = 3 bags

91

No colony-forming unit cells develop from hMSC preparation during 14 day-incubation in HSC-CFU methylcellulose basic media hMSC did not induce tumor formation when injected in NOD/SCID mice both intravenously and subcutaneously during an observation period of more than 90 days

MSC: CLONOGENICITY AND IN-VIVO TUMORIGENESIS

92

MSC: CRITERIA FOR BATCH RELEASE

Phenotype: CD105+: >70% Negative for Mycoplasma Viability >80% by 7-amino-actinomycin D exclusion CD73+: >70% CD90+: >70% CD14+: <10% CD45+: <10% CD34+: <10% Negative for Gram positive and Gram negative bacteria, and fungi Endotoxin below 5 EU/kg confirmed by Trypan blue exclusion

93

These slides are belonging to

Giuseppe Remuzzi, M.D.

Mario Negri Institute for Pharmacological Research, Bergamo, Italy.

Using these slides is only authorized by mentioning the source

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