Engraftment Monitoring after HSCT · 2018-04-01 · Patients with MC level 3 early after HSCT have...

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

Complete Chimerism Mixed Chimerism

RelapseRejection

Donor cell

Normal Recipient cell

Malignant Recipient cellX

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

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

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

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGAVWA

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

Post-BM

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)

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

2 months

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

10-25%

Rejection

Complete Chimerism

patient

Complete Chimerism

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

WBCs CD3+

days after BMT

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

100BU14 - CY200BU14 - CY160BU14 - CY120

210pts

76 pts

26 pts

mMixed Chimerism in Thalassemia after HSCT

2 months 24 months0

20406080

BU14/CY200BU14/CY160BU14/CY120

rejection

210 11/76

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

767/28

2 months 24 months0

100 BU14 - CY200BU14 - CY160BU14 - CY120

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

2 months 24 months

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

20 60 150 400 575 935 1159 1314

Days after BMT

PB BMG. H. BMT 15-12-2005

020406080

60 365 1095 1825

WBCs CD3+ CD19+

days after BMT

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

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

20 60 150 400 575 935 1159 1314

Days after BMT

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

% 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

% 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

MIXED CHIMERISM AFTER BMT IN THALASSEMIA

0 24 48 72 96 120months after BMT

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

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.

60 180 365 720 1085 1450 2170 2890 3100

Days after HSCT

BM PB Hb RBC

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

Th0 Th1 Th2 Tr1

T cell clones

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

Suppression ?

Suppressive activity of host and donor alloreactive Tr1 cell clones

+F28 +F17 +F1 +E10 +F29

75%23%

Patient PBMC + CD3/CD28 mAb

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

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29

Donor clone 30 + CD3/CD28 mAb

45%53%

02000400060008000

49% 55%

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

+F28 +F17 +F1 +E10 +F29

75%23%

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29

45%53%

02000400060008000

49% 55%

+F28 +F17 +F1 +E10 +F29

75%23%

02000400060008000

100001200014000

DON+F14 +F24 +F27+F17 +F29

45%53%

02000400060008000

49% 55%

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

Engraftment Monitoring after HSCT · 2018-04-01 · Patients with MC level 3 early after HSCT have...

Documents

Oncology Models and Services - Taconic Biosciences or scids, or for engraftment of patient-derived tumors. • The best choice for human immune system engraftment mice, with successful

Alterative Donor HSCT

Complete Microbiota Engraftment Is Not Essential …Complete Microbiota Engraftment Is Not Essential for Recovery from Recurrent Clostridium difﬁcile Infection following Fecal Microbiota

Epstein-Barr Virus (EBV) complications following HSCT ... Virus (EBV) complications following HSCT: from viremia to pppyppost transplant lymphoproliferative disease (PTLD) and lymphoma

Prostaglandin E2 enhances engraftment of human cord blood … · 2019-11-26 · Prostaglandin E2 enhances engraftment of human cord blood stem cells and shows long-term safety in

Antifungal prophylaxis of patients undergoing allogenetic ......and improve the OS of patients after allo-HSCT. Keywords: Invasive fungal diseases, Allogenetic, Stem cell, Transplantation,

INTRODUCTION TO HAEMATOPOIETIC STEM CELL TRANSPLANT(HSCT)

Adipocytes and stem cell engraftment

Whole CMV Proteome Pattern Recognition Analysis after HSCT ... · Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the

Improving Umbilical Cord Blood Stem Cell Engraftment by Ex ... · Improving Umbilical Cord Blood Stem Cell Engraftment by Ex Vivo Expansion ... adipocytes and mesenchymal stromal

Allogeneic hematopoietic stem cell transplantation in adult ......hematopoietic stem cell transplantation (HSCT) are unsatisfactory. Therefore, allogeneic (allo) HSCT has been Therefore,

BK Virus infection in HSCT

Infections in HSCT

Managing MF and SS with Allogeneic HSCT - Cutaneous …cutaneouslymphoma.stanford.edu/documents/SY58 allo HSCT in MFSS… · Managing MF and SS with Allogeneic HSCT Youn H Kim, MD

Azacitidine as Maintenance Therapy Following Hematopoietic ...sbtmo2018.com.br/.../04...ANDRE-AMERICO-SEGOVIA-01.pdfIntroduction Azacitidine • Safely administered after HSCT ª º

An Improved Protocol for Efficient Engraftment in NOD

Lymphoma Reporting Basics › Meetings › Materials › CRPDMC... · • Autologous HSCT • Offered to patients with relapsed or primary refractory disease (after platinum-based

Supplementary Materials for...6 Fig. S5: Pulmonary engraftment after organotropic cell transplantation in the humanized herPAP model. (A) Engraftment of hCD45 + donor derived cells

Clinical Utility - DORAK · HLA Matching & Transplantation Success Together with ABO, CMV compatibility and negative xM: Absolute requirement in HSCT engraftment (but GvL?) ... for

2019: Waldenstrom's Macroglobulinemia Pre- HSCT Data