Engraftment Monitoring after HSCT · 2018-04-01 · Patients with MC level 3 early after HSCT have a higher risk of graft rejection Engraftment status at 60 days after HSCT Patients

Engraftment Monitoring after HSCT

Marco AndreaniLaboratory of Immunogenetics and Transplant Biology (LIBT) - IME Foundation

10th InternationalSummer School on Immunogenetics

Stintino | Sardinia | Italy15th - 18th September 2013

Engraftment monitoring is an important issue in HSCT

The early detection of residual host cells represents acrucial information relative to the potential outcome of thetransplant


Patient before HSCT

Patient after HSCT

XX

X


Complete Chimerism Mixed Chimerism

Donor

RelapseRejection

XX

X

Donor cell

Normal Recipient cell

Malignant Recipient cellX

HSCT

Mixed ChimerismDefinitionNatural and inducedMethods commonly used for detectionGraft rejection and relapseImmunological tolerance




Mixed chimerismindicates the presence of both donor and recipientlymphohematopoiesis

Split chimerismindicates the presence within a single compartment ofdifferent donor and recipient cells proportion (example:lymphoid vs myeloid lineages)

Microchimerismindicates the presence of donor cells that are detectableonly with very sensitive techniques




Natural Pregnancy

Induced TransfusionSolid organ and HSCT transplantation


Natural Pregnancy



Naturally acquired Mc derives primarily from maternal cells in her progeny, or cells of fetal origin in women

Both maternal and fetal Mc are detected in hematopoieticcells including T and B cells, monocyte/macrophages, NKcells and granulocytes

Mc appears also to generate cells such as myocytes,hepatocytes, islet cells and neurons

Interactions between a pregnant woman and heracquired microchimerism cell populations may have thepotential to influence normal reproduction and be apositive favorable prognostic factor for preeclampsia

Mold showed that the human fetal immune system takes advantage ofan additional mechanism: the generation of regulatory T cells (Tregs)that suppress fetal immune responses

Natural microchimerism

NIPA e NIMA

HLA-G

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.

Natural Pregnancy



Microchimerism and trasfusion after polytrauma

Induced mixed chimerism

Natural Pregnancy





What is needed for clinically relevant analysis

Panel of genetic loci sufficient to differentiate donors from recipients

Quick turn-around-time

Sensitivity – ability to detect low numbers of cells

Small sample size

Markers commonly used for mixed chimerism detection

- Red blood cell phenotyping- Karyotyping - HLA typing

- FISH analysis

- VNTR - STR (2-5%)- Real Time PCR (0.1 – 0.05%)

Increasing Sensitivity

FISH for chromosome Y ed X

Short tandem repeat (STR) are polymorphic DNA loci thatcontain a repeat nucleotide sequenceThe STR repeat unit can be from 2 to 7 nucleotides inlength

DNA fragments analysis – STRShort tandem repeats

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another


13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGAVWA

AMEL

AMEL

Multiplex PCR• Over 10 Markers Can Be

Copied at Once• Sensitivities to levels less than

1 ng of DNA• Ability to Handle Mixtures

and Degraded Samples• Different Fluorescent Dyes

Used to Distinguish STR Alleles with Overlapping Size Ranges


PRE

Post-BM

DON

Markers commonly used for mixed chimerism detection

- Red blood cell phenotyping- Karyotyping - HLA typing

- FISH analysis

- VNTR - STR (2-5%)- Real Time PCR

Real-Time PCR Chimerism

Sensitivity: Detection of 0.05%of minor component in a mixed DNA sample when starting with 250ng of DNA

Real-Time PCR Chimerism

Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

STANDARDS FOR HISTOCOMPATIBILITY & IMMUNOGENETICS TESTING

Version 5.7



Relapse of the disease

The chimeric status of the patients postallo-HSCT has assumed increasedimportance in addition to thedetermination of MRD analysis withspecific tumor markersMethods to predict disease relapseafter chemotherapy or HSCT allowearly intervention may improve theprobability of long-term disease freesurvival (DFS)

XX

X

They developed a multiplex PCR for use in the simultaneous detection ofhematopoietic chimerism and mutations in nucleophosmin (NPM1) andfms-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD)

Unsorted and lineage-specific donor engraftment were measured in 134patients: 43 patients had an incomplete CD34(+) chimerism with no otherevidence of relapse

Relapse-free survival at 3 years of the 91 patients with stable donor chimerismwas 74% while in the 43 patients with incomplete donor chimerism was 40%

After HSCT with reduced intensity conditioningregimens, high probability to observe mixed chimerism

Therapeutic intervention after HSCT, including DLI

Relapse of the disease

Different Conditioning regimens for HSCT and DLI



Thalassemia represent an unique model in order to study the coexistence of donor and recipient cells after HSCTs due to the

absence of residual malignant host cells

Mixed Chimerism in Thalassemia after HSCT


Transient Mixed Chimerism

0

50

100

2 months

% o

f pts

with

MC

MC - 153 pts



Transient MC represents a risk factor for graft rejection

Number of RHCs present early after HSCT

Presence in the PB of split chimerism within the CD3+ cells

Conditioning regimes used

Transplant outcomeChimerism status early after HSCT

> 25%

10-25%

< 10%

Rejection

PMC

Complete Chimerism

patient

donor

Complete Chimerism

Complete Chimerism

PMC

PMC

Rejection

Rejection


Patients with MC level 3 early after HSCT have a higher risk of graft rejection

Engraftment status at 60 days after HSCTPatients analyzed: 105

N°Pts

Engraftment evolutionMC CC Rej

MC Level 3 (RHCs>25%)

10 2 3 5 (50%)


CC 50 3 46 1 (2%)





Conditioning regimes used

0

20

40

60

80

100

80

WBCs CD3+

days after BMT

% o

f don

or c

ells

UPN: 1023






Conditioning regimens used


Conditioning Regimens

Eradication:- BU 14 mg/Kg

ImmunosuppressionThree different CY doses:

- 200mg/Kg

- 160mg/Kg

- 120mg/Kg

2 months 24 months0

20

40

60

80

100BU14 - CY200BU14 - CY160BU14 - CY120

210pts

76 pts

26 pts

inci

denc

e of

mix

ed c

him

eris

mMixed Chimerism in Thalassemia after HSCT

2 months 24 months0

20406080

100

inci

denc

e of

mix

ed c

him

eris

m

BU14/CY200BU14/CY160BU14/CY120

rejection

210 11/76

28

10/155 2/26 5/56 10/210

767/28


2 months 24 months0

20

40

60

80

100 BU14 - CY200BU14 - CY160BU14 - CY120

inci

denc

e of

mix

ed c

him

eris

m

210pts

76 pts

28 pts

10/155 2/26 5/56


no evolution to graft failureno evolution to complete chimerism

MC is defined persistent when donor and recipient cellscoexist for more than 24 months after HSCT

Persistent Mixed Chimerism

Functional graft

In many cases the proportion of cells of recipient origin is extremely large

020406080

100

2 months 24 months

% M

C


Patients Follow-up (years)

Proportion of donor nucleated

cellsSal A 7 80Tey M 7 46

Sat F 14 25You M 5 29Ven M 17 70Tri A 17 84Kyr A 9 18Fre G 14 40Cel S 10 59Car F 17 20Gan H 7 15


0

20

40

60

80

100

20 60 150 400 575 935 1159 1314

Days after BMT

% D

onor

PB BMG. H. BMT 15-12-2005


020406080

100

60 365 1095 1825

WBCs CD3+ CD19+

days after BMT

% o

f don

orce

lls

UPN 1153


Mostly of the studies reported in literature showed the presence of mixed chimerism in the nucleated cells….

….rather than in the mature erythrocytes, cells functionally crucial for the patients affected by haemoglobinopathies.


Donor engraftment in RBCs


We investigated the presence of MC in thered blood cells by citofluorimetry analysis to detectchimerism in RBC using ABO or Rh differences

ctr C

c D

E e

Recipient: “c” and “E” pos. Donor: “C” and “e” pos


0

20

40

60

80

100

20 60 150 400 575 935 1159 1314

Days after BMT

% D

onor

PB RBC BMG. H. BMT 15-12-2005

RBC: 80% and 73% donor


Other thalassemic patients with PMC

PERSISTEN MIXED CHIMERISM

Patients Follow-up

(years)

% donor nucleated

cells

% donor RBCs

SA 3 72 100AB 4 59 99TM 7 50 100SF 10 25 80GH 3 15 80

Andreani M, Haematologica. 2011 Jan;96(1):128-33.


Patients Follow-up

(years)

% donor nucleated

cells

% donor RBCs

SA 3 72 100AB 4 59 99TM 7 50 100SF 10 25 80GH 3 15 80

PMC in pazienti talassemici dopo TMO

% donor BFU-E

7550552830

• Detection of mixed chimerism in the nucleated cells• Difference between transient and persistent mixed chimerism• The detection of mixed chimerism in red blood cells• Control of the erythroid compartment expansion• Associated between PMC and specific immune tolerance


LATE REJECTION AFTER BMT IN THALASSEMIA

2 6 12 18 24 30 360

20

40

60

80

100

MIXED CHIMERISM AFTER BMT IN THALASSEMIA

0 24 48 72 96 120months after BMT

0

20

40

60

80

100

% o

f don

or e

ngra

ftmen

tevolution of M.C. in 11 patients with RHCs > 25%

• Presence of T regulatory cells

• Donor – Recipient Origin

• Specific suppressor activity


Associated between PMC and specific immune tolerance





Associated between PMC and specific immune tolerance

In the recent years many studies have shown that mixed chimerism isstrongly associated with a state of immunotolerance, particularly in the clinicalexperience of solid organ transplantation.

Sachs DH, Sykes M, Kawai T, Cosimi AB. Immuno-intervention for the induction ofransplantation tolerance through mixed chimerism. Semin Immunol. 2011Jun;23(3):165-73.Mathes DW, Hwang B, Graves SS, Edwards J, Chang J, Storer BE, Butts-MiwongtumT, Sale GE, Nash RA, Storb R. Tolerance to vascularized composite allografts in caninemixed hematopoietic chimeras. Transplantation. 2011 Dec 27;92(12):1301-8.

Sykes M, Immune tolerance: mechanisms and application in clinical transplantation,Journal of internal medicine, 2007 Blackwell Publishing.


Mixed chimerism as an approach to transplantation toleranceThe ability to achieve transplantation tolerance with HCT has been well

documented in patients who first received HCT with conventional myeloablative conditioning to treat a haematological malignancy, and later accepted an organ transplant from the same donor without chronic immunosuppressive therapy.

0

20

40

60

80

100

60 180 365 720 1085 1450 2170 2890 3100

Days after HSCT

% d

onor

BM PB Hb RBC

RBC

PB

BM


Hb Synthesis

Two different cloning of CD4+ cells from a patient with PMC


Serafini G, Andreani M, Testi M et al. Haematologica. 2009 Oct;94(10):1415-26

0

10

20

30

40

50

60

Th0 Th1 Th2 Tr1

T cell clones

% c

ells

1 2 2

5545 46

928 24

3325 28


patient 2nd T cell cloning

patient 1st T cell cloning

normal donor





Host/Donor T cell clones origin (STR analysis)

1st T cell cloning 2nd T cell cloning

ORIGIN n° clones n° Tr1 n° clones n° Tr1

HOST 12 5* 26 7*

DONOR 16 6* 20 6*

TOTAL 28 11 46 13

*Number of Tr1 cell clones from the host/donor T cell clones characterized

Tr1 cell clones of both origins

Both host and donor Tr1 cell clones are present in vivoin the patient with PMC.

Host/Donor T cell clones origin (STR analysis)

1st T cell cloning 2nd T cell cloning

ORIGIN n° clones n° Tr1 n° clones n° Tr1

HOST 12 5* 26 7*

DONOR 16 6* 20 6*

TOTAL 28 11 46 13

*Number of Tr1 cell clones from the host/donor T cell clones characterized

Tr1 cell clones of both origins

Both host and donor Tr1 cell clones are present in vivoin the patient with PMC.



• Specific suppressive activity


Suppressive activity

Recipient

Donor+++++

IFN-gamma production

read – out

Alloreactivity

Adding of Tr1 PMC recipient or donor clones

X

Suppression ?

Suppressive activity of host and donor alloreactive Tr1 cell clones

+F28 +F17 +F1 +E10 +F29

75%23%

IFN

prod

uctio

n

Patient PBMC + CD3/CD28 mAb

HOST

0

10000

20000

30000

40000

50000

0

10000

20000

30000

40000

IFN

prod

uctio

n

68%

27%

Donor PBMC + CD3/CD28 mAb+F28 +F17 +F1 +E10 +F29

HOST

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29

Donor clone 30 + CD3/CD28 mAb

HOST

IFN

prod

uctio

n71%

45%53%

HOST

IFN

prod

uctio

n

02000400060008000

10000

50000

49% 55%

+F17 +E18 +F29 +F13 +R40Host clone 18 + CD3/CD28 mAb

DON


+F28 +F17 +F1 +E10 +F29

75%23%

IFN

prod

uctio

n


HOST

0

10000

20000

30000

40000

50000

0

10000

20000

30000

40000

IFN

prod

uctio

n

68%

27%


HOST

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29


HOST

IFN

prod

uctio

n71%

45%53%

HOST

IFN

prod

uctio

n

02000400060008000

10000

50000

49% 55%


DON


+F28 +F17 +F1 +E10 +F29

75%23%

IFN

prod

uctio

n


HOST

0

10000

20000

30000

40000

50000

0

10000

20000

30000

40000

IFN

prod

uctio

n

68%

27%


HOST

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29


HOST

IFN

prod

uctio

n71%

45%53%

HOST

IFN

prod

uctio

n

02000400060008000

10000

50000

49% 55%


DON

THALASSEMIA PATIENTS AFTER HAPLOIDENTICAL BM HSC THAT DEVELOPED PMC

Higher frequency of IL-10-producing T cell clones in the patient with PMC compared to normal donor:

- are of both donor and host origin

- secrete high amounts of IL-10 compared to Tr1 clones from normal donor

- suppress both host and donor- specific T-cell mediated responses

Serafini G, Andreani M, Testi M et al. Haematologica. 2009 Oct;94(10):1415-26

MORE RECENT ACHIEVEMENTS

Definition of other tolerogenic markers:

Role of Granzyme B

New markers for Tr1 cells

Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- andperforin (PRF)- dependent mechanism that requires HLA class I recognition,CD54/Lymphocyte Function-associated Antigen (LFA)-1 adhesion andactivation via CD2.

High frequency of GZB expressing CD4+ T cells is detected in tolerant patientsand correlates with elevated occurrence of IL-10-producing CD4+ T cells.

The modulatory activities of Tr1 cells are not only due to suppressive cytokinesbut also to specific cell-to-cell interactions which lead to selective killing oftarget cells and possibly bystander suppression

The coexpression of CD49b and LAG-3 enables the isolation of highlysuppressive human Tr1 cells from in vitro anergized cultures and allows thetracking of Tr1 cells in the peripheral blood of subjects who developedtolerance after allogeneic hematopoietic stem cell transplantation.

The use of these markers makes it feasible to track Tr1 cells in vivo andpurify Tr1 cells for cell therapy to induce or restore tolerance in subjects withimmune-mediated diseases.

CONCLUSIONS

Tr1 cells are critically involved in induction and maintenance of PMCafter HSCT in B-Thal pts; however, the mechanisms underlying PMCinduction are still elusive.

The establishment of active tolerance in the lymphoid compartment mightcontribute to the high engraftment of the donor's over recipient's erythroidcells.

Definition of the mechanisms involved in tolerance associated with Tr1cells and PMC will provide tools for designing new protocols for in vivoinduction of tolerance via PMC, and information for reducingpretransplant conditioning regiment, besides contributing to the designgene therapy strategy.

Acknowledgments

HSR TIGET – MilanoSilvia GregoriGiorgia Serafini Chiara MagnaniRosa BacchettaKatharina FleischhauerMaria Grazia Roncarolo

Servizio Trasfusionale S.Orsola BolognaAndrea BontadiniPierluigi TazzariFrancesca Ricci

Centro Trapianti Fondazione IME - RomaGuido LucarelliJavid GazievPietro SodaniKatia PaciaroniFabio TorelliGioia De AngelisCecilia AlfieriCristiano GallucciDomenica SimoneAndrea RovedaLuisa CardarelliMarco MarzialiAntonella Isgrò

ISS - RomaMassimo SanchezValentina Tirelli

Policlinico di Tor Vergata - RomaLidia De FeliceFrancesca AgostiniDaniela Fraboni

LIBT Fondazione IME Manuela TestiMaria TroianoMariarosa BattarraTiziana GalluccioRossella CondelloAnnalisa GuagnanoGiuseppe Testa Chiara StellitanoRenata RosatiAndrea Di LuzioSimona De PetrisEleonora PalladiniMartina Mangione

Acknowledgments

Documents

Engraftment Monitoring after HSCT · 2018-04-01 · Patients with MC level 3 early after HSCT have a higher risk of graft rejection Engraftment status at 60 days after HSCT Patients