UNIVERSITE LIBRE DE BRUXELLES FACULTE DE MEDECINE
Quelle place pour la greffe de cellules souches haploidentiques et comment améliorer son efficacité clinique en manipulant, en post-transplantation, l’environnement cellulaire au moyen de
l’utilisation de populations cellulaires sélectionnées ou de facteurs solubles modulant l’immunité ?
The current place of haplo-identical stem cell transplantation and how to improve its clinical outcome by manipulation of the cellular environment post-transplant using selected cellular
populations or immunomodulatory soluble factors
Thèse présentée en vue de l'obtention du grade de Docteur en Sciences Médicales
PHILIPPE LEWALLE
PROMOTEUR : PROFESSEUR PHILIPPE MARTIAT INSTITUT JULES BORDET
LABORATOIRE D'HEMATOLOGIE EXPERIMENTALE SERVICE D' HEMATOLOGIE
ANNEE ACADEMIQUE 2010-2011
Remerciements
A quelques lieux, témoins privilégiés de mes joies et de mes doutes,
A ce vieux moulin du bord de la Woluwe, observateur fidèle de ma vie étudiante, dont les tours d’ailes ont scandés les premiers chapitres de mon contact avec la médecine et la recherche.
A cet obélisque blanc et majestueux, chroniqueur de mon expérience américaine, qui peut attester de mon apprentissage outre atlantique et de ma découverte de l’immunologie et du nouveau monde.
A cette porte bruxelloise centenaire, rapporteur de mon engagement professionnel, qui peut confirmer ma volonté de travailler sans retenue pour améliorer nos connaissances de l’immunothérapie du cancer.
J’exprime ma gratitude la plus profonde à ma Destinée pour avoir eu la chance de rencontrer au fil des années tant de gens intéressants et enrichissants qui m’ont permis d’évoluer par le partage de leurs connaissances et de leur expérience. Il est bien inutile de les citer car chacun d’eux sait la place particulière et spécifique qu’il occupe sur mon chemin.
Quelques remerciements particuliers vont néanmoins vers
Philippe, pour m’avoir toujours soutenu et encouragé, sans réserve, sans calcul ni bénéfice, pour moi-même, avec le seul but de me permettre de m’épanouir et de rencontrer mes objectifs personnels.
Jean-Luc, pour m’avoir appris qu’il est plus difficile à un médecin de faire de la science qu’à un chameau de passer par le chas d’une aiguille.
John, pour m’avoir communiqué son enthousiasme pour l’immunothérapie et pour m’avoir démontré, que même à notre époque de rentabilité immédiate, l’on pouvait être un excellent professionnel tout en restant un homme de Culture et d’Humanisme.
Nancy, pour m’avoir aidé dans les moments difficiles de l’adaptation à la vie américaine, pour être restée un infaillible support dans les moments de découragement et un aiguillon indispensable pour mener le rêve à son terme.
Redouane, pour avoir, au risque de se perdre, toujours partagé avec moi la folie que rien de grand ne se réalise sans chimère.
Dominique, qui dans une situation complexe, m’a permis de poursuivre l’aventure et m’a fortifié de son soutien et de son intérêt.
La science ne sert guère qu'à nous donner une idée de l'étendue de notre ignorance.
Félicité de Lamennais
Composition du Jury de thèse
Promoteur Présidente du Jury
Prof. P. MARTIAT Prof. J. RASSCHAERT
Laboratoire facultaire Laboratoire de Chimie Biologique d’hématologie expérimentale Université Libre de Bruxelles (ULB) Université Libre de Bruxelles (ULB) Hôpital Universitaire Erasme
Institut Jules Bordet Bruxelles, Belgique
Bruxelles, Belgique
Membres du Jury
Prof. C. BLANPAIN
Institut de Recherche Interdisciplinaire
en Biologie Humaine et Moléculaire (IRIBHM)
Université Libre de Bruxelles (ULB)
Hôpital Universitaire Erasme Bruxelles, Belgique
Prof . A. LE MOINE
Institut d’Immunologie Médicale Université Libre de Bruxelles (ULB) Gosselies, Belgique
Prof. M. TOUNGOUZ NVESSIGNSKY Laboratoire de Thérapie Cellulaire Clinique Université Libre de Bruxelles (ULB)
Hôpital Universitaire Erasme Bruxelles, Belgique
Experts extérieurs
Prof. F. BARON
Département d’Hématologie clinique
Université de Liège (ULg)
CHU de Liège
Liège, Belgique
Prof. J.H. FALKENBURG Department of Hematology Leiden University Medical Center Leiden, The Netherlands
Résumé
Dans la majorité des situations, le système immunitaire autologue est incapable d’éradiquer les cellules leucémiques résiduelles qui échappent à la radiothérapie et à la chimiothérapie, cependant un équilibre peut s’établir entre les cellules leucémiques et immunitaires aboutissant à une rémission pouvant durer plusieurs mois ou années. Si cet équilibre se rompt, une rechute clinique peut se déclarer. Dans ce contexte, il est prouvé que la greffe allogénique de cellules souches hématopoïétiques est le moyen le plus efficace de renforcer les réactions immunitaires contre la leucémie par la réaction du greffon contre la leucémie et ainsi d’obtenir une éradication définitive de la maladie résiduelle chez un nombre significatif de patients. En effet, le concept global de l’allogreffe de cellules souches hématopoïétiques a évolué du concept de transplantation d’organe (remplacement d’un organe malade par un nouvel organe sain) vers celui de créer une extraordinaire plateforme d’immunothérapie à travers laquelle le système immunitaire du donneur contribue à l’éradication des cellules leucémiques persistantes. Donc, la problématique reste celle de trouver les meilleures modalités d’immunomodulation pour achever une prise du greffon, un effet anti-leucémique puissant du greffon, et l’absence ou un minimum d’effet du greffon contre l’hôte. Différentes stratégies existent pour atteindre cet objectif, comme l’utilisation de cytokines pour moduler la reconstitution immunitaire, des déplétions cellulaires globales ou spécifiques du greffon et l’infusion de cellules immunes
«globales» ou spécifiques du donneur après greffe. Ces stratégies sont encore largement à l’étude. Néanmoins, la persistance d’un taux de rechute élevé observé chez les patients leucémiques, après allogreffe reste la cause principale de décès, avant celle liée à la toxicité de la greffe. De plus, étant donné que seulement environ 40 à 70% (dépendant de l’origine ethnique) des patients avec une hémopathie à haut risque, éligibles pour une greffe allogénique, ont un donneur familial ou non familial complètement HLA compatible, des efforts importants ont été développés pour rendre faisable l’utilisation de donneurs familiaux alternatifs, haploidentiques. L’avantage de cette approche est l’accès immédiat à un donneur pour quasiment tous les patients.
Le but du travail décrit dans cette thèse a été l’implémentation d’une stratégie d’allogreffe utilisant un donneur haploidentique. Le travail vise également à développer de façon plus large
des stratégies qui peuvent améliorer le taux de survie sans rechute, non seulement dans le contexte des greffes haploidentiques, mais également dans le cadre des greffes allogéniques en général, ainsi que dans les situations autologues : premièrement, par la manipulation immunitaire non spécifique après greffe et ensuite par le développement de stratégies spécifiques dirigées contre des antigènes leucémiques. En particulier dans la situation allogénique, le but a été d’augmenter l’effet du greffon contre la leucémie sans induire ou aggraver l’effet délétère du greffon contre l’hôte. La première partie de la thèse décrit les résultats cliniques de notre propre protocole de greffe haploidentique, qui a consisté en trois études consécutives de phase I/II. Dans ces études, nous avons voulu déterminer la faisabilité de réaliser des infusions prophylactiques de lymphocytes du donneur après transplantation, et l’impact du remplacement du « granulocyte colony-stimulating factor » (G-CSF), largement utilisé pour permettre une récupération en polynucléaires neutrophiles plus rapide, par du
« granulocyte-macrophage colony-stimulating factor » (GM-CSF), lequel est connu pour ses propriétés immunomodulatrices différentes. La reconstitution immunitaire très lente après greffe haploidentique est majoritairement responsable de l’incidence élevée de décès par infections virales et fungiques précoces, et très probablement des rechutes précoces. C’est pourquoi nous avons cherché à accélérer et à renforcer la reconstitution immunitaire post- greffe sans augmenter la fréquence de réaction du greffon contre l’hôte. Nous avons donc étudié l’impact de l’administration de facteurs de croissance et l’infusion de lymphocytes non sélectionnés du donneur en post greffe haploidentique. Nous avons également implémenté dans notre centre, la génération et l’infusion de lymphocytes T spécifiques anti-cytomégalovirus (CMV) afin d’améliorer la reconstitution immunitaire anti-CMV. D’autre part, nos résultats ont été regroupés dans une étude multicentrique menée par le groupe européen de transplantation de moelle osseuse (EBMT), ce qui nous a permis de comparer nos résultats avec ceux de l’entièreté du groupe. Nous avons parallèlement participé à la conception d’une étude actuellement en cours ayant pour but d’améliorer la reconstitution immunitaire après greffe par la déplétion sélective du greffon en lymphocytes T alloréactifs et par l’infusion après greffe de lymphocytes T du donneur également sélectivement déplétés en lymphocytes T alloréactifs.
Afin d’optimaliser l’effet anti-leucémique du système immunitaire, nous avons débuté un protocole de vaccination par cellules dendritiques (DCs). Ces cellules dendritiques étaient chargées en lysat de blastes leucémiques dans le cas de patients présentant au diagnostic une leucémie aigue surexprimant l’oncogène 1 de la tumeur de Wilms (WT1). Néanmoins dans nos
travaux de génération et d’expansion ex-vivo de lymphocytes T spécifiques de l’antigène WT1, utilisant les DCs de grade clinique, générées et maturées en poches, nous avons rencontré des résultats inconsistants, comme c’était le cas dans la majorité des protocoles cliniques internationaux de vaccination. Nous nous sommes alors posé la question de la fonctionnalité globale de ces cellules et nous avons entrepris une analyse comparative poussée des DCs générées et différenciées en poches ou en plaques. Les DCs générées en plaques sont celles utilisées dans la plupart des travaux précliniques. Cette analyse nous a montré que l’on ne pouvait pas directement transposer les résultats précliniques basés sur des DCs générées en plaques dans des protocoles cliniques basés sur des DCs générées en poches, car ces dernières présentent des déficits fonctionnels importants. Nous avons appris que si l’on voulait utiliser un vaccin à base de cellules dendritiques pour améliorer l’effet du greffon contre la leucémie dans les greffes allogéniques, nous devions être très attentifs quant au protocole utilisé pour la génération de ces vaccins cellulaires. Pour améliorer les approches immunothérapeutiques, la connaissance des mécanismes qui établissent la tolérance tumorale et des façons de manipuler ceux-ci, est critique dans le développement de nouveaux protocoles efficaces. C’est pourquoi nous nous concentrons actuellement sur les conditions nécessaires à l’obtention in vivo d’une réaction immune anti-leucémique efficace lors de l’utilisation d’un produit cellulaire manipulé ex vivo. Plus spécifiquement, nous analysons l’impact de la déplétion en lymphocytes T régulateurs (Tregs) sur la réponse anti-leucémique. Ce travail préclinique a pour but d’améliorer le devenir de patients leucémiques qui ont rechutés et ont été mis en seconde rémission, ainsi que de diminuer le taux de rechute après allogreffe, spécifiquement après greffe haploidentique.
En conclusion, la transplantation haploidentique est actuellement un outil précieux pour de nombreux patients. Les résultats sont au minimum similaires à ceux qui sont obtenus par les greffes non-familiales HLA identiques lorsqu’elles sont pratiquées dans les mêmes groupes de patients. L’immunomodulation spécifique après greffe peut affecter des événements comme la réaction du greffon contre l’hôte et la réaction du greffon contre la leucémie, mais en pratique clinique nous en sommes encore au niveau de la manipulation aspécifique. Nous espérons que les travaux précliniques actuels vont nous permettre d’appliquer des stratégies spécifiques et d’obtenir une manipulation immune anti-leucémique qui aura une influence favorable significative sur le devenir des patients.
Summary
Currently, in most situations, the autologous immune system is unable to eradicate the residual leukemic burden persisting after chemo-radiotherapy, but a balance can be established between leukemic and immune cells leading to a clinical remission for several months or years. If this balance is broken, a clinical relapse can occur. The high incidence of relapses in human cancers demonstrates the frequent inefficacy of the immune system to control these residual cells. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has been proven to be the most effective way to reinforce the immune reaction against leukemia, graft- versus-leukemia (GVL) effect and, so, achieve a definitive eradication of the residual disease in a significant proportion of patients. Indeed, the whole concept of HSCT evolved from an organ transplant concept (to replace a defective ill organ with a new healthy one) to the concept of creating an extraordinary immunotherapeutic platform in which the donor immune system contributes to the eradication of the residual leukemic cells. Thus, the past and present issues remain those of finding the best immunomodulatory modalities to achieve a full engraftment, a powerful GVL effect and no or moderate graft-versus-host disease (GVHD). Different ways to reach this goal, such as post transplant cytokine modulation, specific or global cellular depletion of the graft and post transplant global or specific donor immune cell add-backs, are still extensively studied. Nevertheless, the persistent high relapse rate (RR) observed in leukemia patients after HSCT remains the most important cause of death before transplant- related toxicities. Moreover, since only about 40 to 70% (depending on the ethnic context) of patients with high-risk hematological malignancies, eligible for allogeneic HSCT, have a fully HLA-matched sibling or matched unrelated donor (MUD), a great deal of effort has been invested to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients.
The aim of the work described in this thesis has been to implement a strategy to transplant a patient using a HLA haploidentical donor. The strategy is to try to improve DFS that could be applied both in the autologous or allogeneic context: first, by using nonspecific immune manipulation post transplant and then, by developing specific strategies directed against leukemia antigens. Particularly in the allogeneic situation, the aim was to increase the GVL effect without inducing or aggravating the deleterious GVHD. The first part of this thesis
described our own clinical results, consisting of three consecutive phase I/II studies, in which we tried to determine the feasibility of giving prophylactic donor lymphocyte infusions (DLI) post transplant and the effect of replacing granulocyte colony-stimulating factor (G-CSF), typically used to speed up neutrophil recovery, with granulocyte macrophage colony- stimulating factor (GM-CSF), which is known for its immunomodulatory properties. The slow immune reconstitution in haploidentical transplant is chiefly responsible for the high incidence of early lethal viral and fungal infections, and most probably for early relapses; therefore, we sought to accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. Thus, we have studied the impact of post transplant growth factor administration and of unselected DLI in haploidentical transplant. We have also implemented, in our center, anti-cytomegalovirus (CMV) specific T cell generation and infusion to improve anti-CMV immune reconstitution. Since then, our results have been pooled in a multi-center analysis performed by the European Bone Marrow Transplantation group (EBMT) allowing us to compare our results with those of the entire group. We have also participated in the design of an ongoing study aimed at selectively depleting the graft from alloreactive T cells, and improving post transplant T cell add-backs. In our attempts to generate and expand ex vivo lymphocytes (directed against pathogens (CMV) and leukemia-associated antigens, Wilms' tumor gene 1 (WT1) and to use them in vivo, we found inconsistent results (in the case of WT1) using classical clinical grade dendritic cells (DC) generated and matured in bags, as was the case for the majority of the teams worldwide. This led us to question the full functionality of these DC and we undertook a thorough comparative analysis of DC generated and differentiated in bags and in plates (typical for most pre-clinical studies). This analysis showed us that one cannot transpose pre-clinical studies (using culture plates) directly to clinical protocols (generally using clinical grade culture bags) and that DC generated in bags are functionally deficient. We learned that, if we want to use a DC vaccine to improve the GVL effect in haploidentical transplant, we will have to be careful about the technique by which they are generated. To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to manipulate them is critical in the development of new effective immunotherapeutic clinical trials. This is why we currently focus on how to obtain effective in vivo anti-leukemia immune reactions using an ex-vivo manipulated product to trigger the immunotherapeutic response. More specifically, we are analyzing the impact of regulatory T cell (Tregs) depletion and function for an adequate anti-
leukemic immune response. This pre-clinical work aims at improving the outcome of leukemia patients who have relapsed and been put back into second remission and at decreasing the RR after HSCT, especially in the field of haploidentical transplantation.
In conclusion, haploidentical transplantation has become a valuable tool. The results are at least similar to those obtained using MUD when performed in the same group of patients. Specific immunomodulation post transplant can affect events such as GVHD and GVL, but clinically we are still at the level of nonspecific manipulations. It is our hope that ongoing pre-clinical work will enable us to perform specific anti-pathogen and anti-leukemia immune manipulation that will favorably influence the patient outcome.
Introduction
There is a growing body of evidence that the clinical behavior of tumors relies on genetic changes in malignant cells as well as on their interaction with their environment (stroma, vascularization and immune infiltrate). Therefore, the role of the immune system in the detection and elimination of cancer has gained major interest. The last decades have been marked by numerous clinical vaccination trials, most of them having produced rather disappointing clinical results. Meanwhile, fundamental investigations have told us that achieving a successful immunotherapy is a much more complex process than we thought a decade ago. They have mainly led to a better understanding of the mechanisms responsible for the lack of effective anti-tumor immune response. However, they have also clearly established the concept that the immune system can help to achieve a definitive cure in cancer patients.
Recent data strongly suggest that the success of radiochemotherapy depends on the so-called
“immunogenic death” induced by certain drugs or by radiotherapy1. In addition, different groups are studying the role of the immune system as part of the tumor dormancy theory.
Currently they suggest that in most circumstances, the autologous immune system is unable to eradicate the residual disease achieved after chemoradiotherapy but a balance can be established between tumor and immune cells, leading to a clinical remission for several months or years. If this balance is perturbed, a clinical relapse can occur2. The high incidence of relapse in human cancers demonstrates the failure of the immune system to control these residual cells naturally. In this context, allogeneic hematopoietic stem cell transplantation (HSCT) has proven the most effective way to reinforce the immune action against the tumor (the graft-versus-tumor (GVT) effect or, in the case of leukemia, GVL effect) and achieve a definitive eradication of the residual disease in a significant proportion of patients, especially in hematological malignancies. Nevertheless, the high RR observed in leukemia patients after HSCT remains the most important cause of death exceeding transplant-related mortality (TRM). Tumors develop many ways to escape immune surveillance and favor their own growth. Even when a potentially potent immune response exists (which can be demonstrated in vitro), tumors induce regulatory, suppressive and inhibitory mechanisms to protect themselves from this immune response. My first work on detection of specific anti-tumor lymphocytes concluded that it was not possible to detect any significant frequency of anti-tumor helper T
cells in the lymphocyte population of chronic myeloid leukemia (CML) patients3. Since then, the role of Tregs in inhibiting the anti-tumor immune response has been unveiled and characterized. This has given us a hint about why we failed to detect a functional anti-leukemic response in the peripheral blood mononuclear cells (PBMC) population of these patients. This mechanism has now been confirmed by experiments showing that high affinity ex vivo generated anti-tumor cytotoxic T lymphocytes (CTL) work better if they are given to patients after heavily immunosuppressive chemotherapy, also removing the Tregs population. This novel concept implies the necessity of eliminating the Tregs to allow effective CTL anti-tumor activity. In addition to Treg depletion, the lymphocyte homeostasis concept also explains the facilitation of these new tumor-specific lymphocytes expansion after intensive immune suppression (lymphodepletion). These data indicate a need to develop newer approaches for therapeutic improvements. These improvements should be implemented in new autologous immunotherapeutic approaches to increase the rate of sustained complete remissions as well as after HSCT, which is currently the most effective, albeit non specific, cellular immunotherapy in leukemia. Compared to the autologous approach, allogeneic HSCT already has a stronger impact on the immune system of the patient. It offers a unique opportunity for the expansion of a new immune system that, in theory, is not tolerized to the tumor cells. The conditioning regimen, necessary for the engraftment of the donor cells, creates an immunological vacuum that can be used for the expansion of tumor-specific lymphocytes. Moreover, allogeneic transplantation has the unique feature of potentially developing GVT not restricted to tumor- associated antigens (TAA) but also directed against tissue-restricted polymorphic minor histocompatibility antigens (mHags) differing between donor and recipient. The whole concept of HSCT has evolved from the organ transplant concept of replacing a defective organ with a healthy one to the concept of creating an extraordinary immunotherapeutic platform upon which one can build new and more efficient post-remission immunotherapies in leukemia4 (Figure 1). The initial conception of the transplant conditioning regimen as a radical anti- leukemia chemo-radiotherapy devised to destroy the ultimate residual leukemic cell before replacing the marrow by a healthy one has since evolved to a new concept. This new concept consists of finding the best immunomodulatory strategies to achieve full engraftment, a powerful GVT effect and minimal GVHD5-7. Different ways to reach this goal by using post transplant cytokines, specific or global lymphocyte depletion of the graft or post transplant global or specific donor immune cell add-backs are being extensively studied. Another
approach is to reduce or even remove the myeloablative part of the regimen responsible for severe toxicities, using reduced intensity conditioning (RIC), thereby providing the patient in complete remission (or with minimal residual disease) with only the immunotherapeutic benefits of the allogeneic transplant8. Today HSCT can be viewed as the first large-scale successful cellular immunotherapeutic treatment. Nevertheless, rapid and complete immune recovery after allogeneic HSCT in order to increase the rate of long-term DFS is still an unmet goal. The major objective should be to improve on the "natural" immune reconstitution by increasing its specific anti-leukemic activity, thereby rendering HSCT a good option for very high risk or chemorefractory patients for whom allotransplant is still largely unsuccessful even with intensive myeloablative conditioning. Since only about 40% to 70% (depending on the ethnic context) of patients eligible for allogeneic HSCT have a fully HLA-matched donor, either sibling or MUD, a great deal of effort has been expended to make the use of an alternative haploidentical sibling donor feasible. The advantage of this procedure is the immediate availability of a donor for almost all patients. This reduces the risk of relapse/death for patients lacking a sibling donor, in which case, the search for a fully MUD may take months and then often can turn out to be unsuccessful. This is especially true for patients of some ethnic minorities, which are poorly represented in public donor registries.
In the 1950s, a consensus formed on the acceptance and the adjustment of Burnet’s seminal idea of tolerance and rejection mechanisms. According to this consensus, the proper criterion of immunogenicity was the discrimination between self and non-self. The central mechanism of any immune system was the recognition of what is foreign: no element that distinctively belonged to the organism triggered an immune response whereas every single foreign element did. More recent experimental data have questioned the self/non-self theory and a new concept based on the so-called "immunogenic continuity" has emerged. It allows us to better understand how a new antigen is integrated among the usual self antigens of the organism without inducing an effective immune response. This induction of tolerance for a new antigen occurs during repeated contacts under non-immunogenic conditions. This "continuous"
tolerance implies activation of the regulatory components of the immune system. The application of this new concept in the context of allogeneic HSCT has allowed for the emergence of haploidentical transplantation. Currently this type of transplant has become an important alternative for people lacking a full genotypically matched donor. Moreover, the
demonstration that in haploidentical HSCT, the GVL effect can be reinforced by the donor's natural killer (NK) cells in the presence of inhibitory killer cell immunoglobulin-like receptor (KIR) mismatch in the GVH direction, is likely to make this option, which used to be the last resort, the first choice for high risk patients. Many efforts have been made to develop new conditioning regimens, to optimize the graft processing and to improve post transplant immune recovery. Slow immune reconstitution is largely responsible for the high incidence of TRM in haploidentical transplants, a consequence of the high incidence of early viral and fungal infections. Improvements in haploidentical transplant require strategies that accelerate and strengthen the post transplant immune reconstitution without increasing the GVHD rate. In fact, I have studied the impact of both post transplant growth factor administration and unselected DLI in haploidentical transplant. We also participated in the design of an ongoing study aimed at selectively depleting the graft of alloreactive T cells and improving post transplant T cell add-backs. In our center, we have also implemented CMV-specific T cell generation to improve immune reconstitution by using CMV-specific T lymphocyte infusions.
To improve immunotherapeutic approaches, the understanding of the mechanisms underlying tumor tolerance and how to override them are of major importance in developing new successful immunotherapeutic clinical trials. We are currently focused on obtaining effective in vivo anti-leukemia immune responses by using an ex-vivo manipulated product to trigger an immunotherapeutic response. More specifically, we are analyzing the impact of Treg depletion and function on anti-leukemic immune responses. This pre-clinical work is targeted at improving the outcome of leukemia patients who have relapsed and been put back into second remission as well as decreasing the RR after HSCT particularly in the field of haploidentical transplantation.
Theoretical Background
1. Allogeneic stem cell transplantation
Achieving a cure for acute myeloid (AML) or lymphoblastic (ALL) leukemia remains a challenge. While more than 70% of young adult patients will enter a first complete remission (CR1) after induction chemotherapy, a substantial number experience disease relapse9. Allogeneic HSCT is a curative treatment option for some patients. But only one-third of the patients have a matched sibling donor. The probability of finding a fully MUD ranges from less than 10% in ethnic minorities to 60%-70% in Caucasians. Several studies have shown that the results of MUD transplants could approach those of transplant using a HLA compatible sibling donor10-12. An alternative stem cell source is umbilical cord blood (UCB). Initial UCB transplants were limited by the minimum cell doses required for adult transplant, but more recently, the use of double cord transplants has shown efficacy, lowering the risk of graft rejection and possibly the risk of relapse as well13-15. Still, for HSCT, using bone marrow (BM) and peripheral blood stem cells (PBSC), the EBMT group reports a six year leukemia-free survival (LFS) of 55±1% for AML patients transplanted in CR1 with a matched sibling donor compared to 46± 3% for MUD transplants and 44±3% versus 37±4% for patients transplanted in second complete remission (CR2) (EBMT hand book 5th edition 2008). In all groups, only patient age and disease status markedly impacted on TRM. The effect of the major histocompatibility complex (MHC) and non-HLA immunogenetics on allogeneic transplant outcome is briefly explained in Appendix 1. For AML and ALL, the current guidelines for allogeneic transplant indications are based on risk stratification by karyotype and molecular biology. We will expand this topic using the example of AML. The current consensus for AML is based on cytogenetic stratification into good, intermediate, and poor-risk AML (Table 1).
Patients with good-risk AML in CR1 are advised to undergo consolidation chemotherapy with autologous HSCT considered an acceptable alternative. The recommendation for patients with poor-risk AML in CR1 is to undergo allogeneic HSCT. Currently there is no preferred therapy for patients with intermediate-risk AML in CR1; allogeneic HSCT, consolidation chemotherapy and autologous HSCT are considered equally good options. This might change
with recent report allowing better stratification in this group16. Indeed, a significant proportion of patients relapsing after an autologous HSCT consolidation can still benefit from an allogeneic HSCT after achieving a second remission. However, a recent systemic review and meta-analysis of prospective trials evaluating allogeneic HSCT versus autologous HSCT therapies for AML in CR1 concluded that there was a significant benefit for allotransplant in the intermediate risk group17. According to this meta-analysis, we might anticipate a 5-year overall survival rate for AML in CR1 in the control (autologous SCT) group of approximately 45% for intermediate risk and 20% for poor risk AML, respectively, and for patients assigned to undergo allogeneic HSCT, 52% and 31% respectively. In this review, patients were eligible up to 60 years of age in individual studies and the conditioning regimen was myeloablative;
therefore, it remains unclear if older eligible patients obtained an equivalent benefit; also, the impact of co-morbidities was not assessed. The nature of AML changes as patient age increases. Over age 55, AML becomes increasingly difficult to treat. In addition to a more unfavorable disease profile, patients over 55 more often have a diminished performance status (PS) and an increased number of co-morbidities, both of which reduce the likelihood of a favorable short-term (remission rate) and long-term LFS outcome. Patients over 55 have a 45%
chance of CR after induction versus 70% for younger patients. In the mid-1990s, a new era opened: the RIC allotransplant aimed at minimizing the TRM (related to drug toxicity, which increases with age and/or co-morbidities) and infections while preserving the GVL effect. Its emergence has opened up new possibilities for older AML patients in CR. Recent studies suggest a better outcome for older AML patients treated with RIC HSCT than for those treated with conventional chemotherapy. Nevertheless, despite its wide use, to date, there have been no prospective comparative studies. Data come from phase II clinical trials and retrospective analyses. In addition, the conventional outcome measures in allogeneic transplantation such as day +100 mortality, became irrelevant in the era of RIC due to reduction in early regimen- related toxicities. RIC HSCT did not always translate into improved overall survival due to late attritions from relapse, GVHD or late infectious complications. The enthusiasm for performing RIC in malignant diseases seems to have reached a plateau but its true potential probably remains unexplored18;19. Currently, the strongest argument in favor of RIC transplants is the opportunity to give an allogeneic HSCT option to a category of patients who otherwise would not be eligible20-22 (Figure 2). The difficulty with the current cytogenetic stratification used to define risk groups is that most patients fall in the intermediate risk group and 50% of them
have a normal karyotype. Thus, the intermediate group actually includes a very heterogeneous population. As a result, there remains a need to individualize the allogeneic HSCT decision further, based on factors like age, co-morbidity and the presence of additional molecular lesions. To fully improve the decisional tree, we must also take into account the balance between the risks taken by transplantation in CR1 compared to the chance of being rescued by a transplant in CR2. In this intermediate-risk group, 45% of the patients can be cured without allogeneic HSCT (the TRM is about 10% and the RR 45%). About 40% of the relapsing patients can achieve a CR2 and about 44% of the patients transplanted in CR2 stay disease- free, which means that roughly 10% of the patients who were not transplanted in CR1 can be cured by a second-line HSCT23. In other words, the proportion of patients cured by a CR2 transplant strategy is 53% compared to 52% of patients cured by the CR1 transplant option.
Therefore, the two strategies show similar results in terms of DFS in intermediate-risk patients.
Consequently, in this category, the decision to transplant in CR1 versus waiting until first relapse is particularly difficult24. This dilemma confirms the need for a better stratification of the patients included in the intermediate group to segregate the 45% who do not need an allogeneic HSCT from the 45% who need it in CR1. Cytogenetic and molecular risk profiling in AML is an evolving field and can help to make more appropriate decisions within a given cytogenetic risk group. In the future, the analysis of the immune environment may also improve individualized stratification but this is a field still largely unexplored. There are already major advances in molecular tools with potential application for this purpose. For instance, Schlenk et al. reported that for patients with cytogenetically normal AML (who would be classified as intermediate-risk), allogeneic HSCT was beneficial for those with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) or, in the absence of FLT3-ITD, for those without mutations in the nucleophosmin (NPM1) gene or the CCAAT/enhancer-binding protein alpha (CEBPA) gene. For those who have mutations in NPM1 and without FLT3-ITD there was no apparent benefit to have a matched sibling transplant; except when there was an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) mutation which still constitutes a poor prognostic factor for this subgroup of patients25-27. C-kit mutation also appears to be a poor prognostic factor even in otherwise good-risk AML. However, such novel genetic lesions, as well as whole genome analyses, RNA and micro-RNA profiles that have the potential to further refine AML risk are not yet in routine clinical use28-30. Minimal residual disease monitoring by PCR-based assays can also be a useful tool to identify those in CR1 with
an inadequate response who would benefit from an allogeneic transplant upfront, while those with a good molecular response could be spared from it. As an example, overexpression of WT1 occurs in more than 80% of AML patients31. WT1 molecular positivity after induction chemotherapy is highly predictive of relapse and confirms the need for an allogeneic transplant32. These decisions must take into account as well individual risk factors. If the decision is made to delay transplantation until first relapse, physicians should ensure that salvage transplantation is possible, including identifying the source of hematopoietic stem cells in advance of relapse and developing a careful monitoring plan for the patient while in first remission. Only 40% of the patients who relapse achieve CR2; their DFS at 3 years after chemotherapy is 31% when the relapse occurs after 12 months and 0% if the relapse occurs during the first 12 months compared to an overall 44% for the allogeneic transplant option.
Regardless, the transplant option is highly recommended for all patients in CR2. However, there is no clear evidence that early relapsing patients should have a new induction therapy to reach CR2 before moving to a myeloablative allotransplant when the sibling donor is immediately available; thus both approaches seem sensible and show similar results with about 18% long term survivors24. For well defined high risk patients, particularly in patients over 55 years, we must develop treatment strategies to improve the initial complete response rate using novel agents, radioimmunotherapy or combinations; we have also to find a way to increase allogeneic HSCT efficiency even for patients who do not achieve CR. We also need readily available alternative stem cell sources for those who definitely need a transplant in CR1. The average duration of complete remission with conventional chemotherapy for AML patients older than 60 years is only around six months33 and it generally requires, on average, about three months to identify an unrelated donor and proceed to transplantation. Thus, if initial typing is delayed until after a complete remission is achieved, as many as 50% of patients may relapse before they can be transplanted34. In this situation, a haploidentical sibling donor as an alternative stem cell source could be used early in CR1 if one can find a conditioning regimen adapted to these older patients.
2. Haploidentical transplantation
In the last 15 years, haploidentical transplant has become a clinical reality for adult patients lacking an HLA-identical donor35-40. The major stimulus for developing this transplantation approach comes from donor availability. Virtually all patients have a haploidentical family member who potentially can be a suitable donor. This donor can be immediately identified and the graft collection organized rapidly. Often several potential haploidentical donors are identified and a selection based on age, sex, infectious disease status or blood group is possible.
Moreover, a donor with GVH NK alloreactivity, based on KIR mismatch in the donor-recipient pair may readily be selected. Currently, haploidentical transplant with a favorable KIR mismatch might even be considered the best choice for patients with a bad prognosis AML considering the lower RR in this case41-43. The choice of a G-CSF mobilized peripheral blood progenitor cell collection allows for control of the graft composition and the choice of a sibling offers immediate access to donor-derived cellular therapies if required after transplantation.
Furthermore, for nearly all patients, a second donor or a second graft from the same donor would be available in the rare instances of graft rejection. Nonetheless, this type of transplant is still regarded as an experimental procedure by many hematologists; consequently, patients are often offered this type of transplant too late in the disease course or as a last resort despite more than a few encouraging reports. Significantly, recent recommendations to transplant earlier in less heavily pretreated patients have been associated with a reduction in TRM19;44.
2.1. Clinical results in haploidentical transplantation
Haploidentical transplant modalities are based mainly on a high-intensity conditioning regimen but RIC has also been used. Two general strategies are under study depending on whether in vitro T cell depletion (TCD) is used. RIC transplantations are usually based on an unmanipulated graft or partially depleted graft with the need of post transplant immunosuppression. On the other hand, the myeloablative approach is largely based on extensive TCD and infusion of megadoses (≥ 10 x 106/kg) of CD34-positive (CD34+) stem cells without post transplant immunosuppressive drugs.
2.1.1. T cell depleted graft 2.1.1.1. Partial T cell depletion
The first results with partial ex vivo TCD showed encouraging results in terms of GVHD, with a median T cell dose in the graft of 5x104/kg, the use of anti-thymocyte globulin (ATG) in the conditioning regimen along with cyclosporine (CSP) and steroids as post transplant acute GVHD (aGVHD) prophylaxis. The incidence of aGVHD was 13% and for chronic GVHD (cGVHD), 15%. However, the 5 year overall survival was only 19% because of a high 5 year cumulative incidence of relapse (31%) and TRM (51%) related to the use of post transplant immune suppression. The Tubingen group developed a strategy based on CD3/CD19 depletion.
The median T cell dose in the graft was 4.4x104/kg, (range, 0.6x10-2 to 4.4x105 CD3+ T cells/kg) but the number of NK cells, monocytes and other antigen presenting cells (APC) that have engraftment facilitating properties was higher. The patients received a median CD34+
stem cell dose of 7.6x106/kg. The engraftment was rapid in all patients and demonstrated that successful haploidentical transplant may be feasible even without mega doses of CD34+ stem cells; but the price to pay for this higher dose of lymphocytes is a higher and more severe GVHD rate. A reduced conditioning regimen was used including fludarabine (FLU), thiotepa, melphalan and OKT3; there was also no post transplant aGVHD prophylaxis. This resulted in a high incidence (48%) of GVHD, grade II-IV. TRM was 20% and the one year DFS was 31%
45.
2.1.1.2. Extensive T cell depletion with “megadose” stem cells
The increasing interest in haploidentical transplantation was generated by the pioneering work of Reisner et al. and the initial 1994 report from the Perugia group about the use of more intensive conditioning followed by transplantation of a “mega dose” of TCD CD34+ stem cells harvested from PBMC46. The median cell numbers in the Aversa et al. report were 12.8x106/kg for CD34+ stem cells and 1x104/kg for CD3+ T lymphocytes. The conditioning regimen included total body irradiation (TBI) and ATG with no use of post transplant immunosupression to prevent GVHD. Updated results of the Perugia group show an engraftment rate of 95% and an incidence of aGVHD (grade II-IV) of 5%; the TRM was 36%
for patients transplanted in any CR and 58% for patients transplanted in relapse47. Most deaths resulted from infection mostly due to CMV and Aspergillus. Only 18% AML and 30% ALL
patients transplanted in any CR relapsed. Moreover, the RR was significantly lower after transplantation from NK alloreactive donors for AML, 3% versus 47%. ALL and AML patients together, in any CR, receiving a transplant from an NK cell alloreactive donor had 67% event- free survival (EFS) versus 18% for patients transplanted from non-NK alloreactive donors. The overall survival rate for AML patients, regardless of NK alloreactivity, was 55% (CR1) and 33% (CR2) and 28% for ALL patients (any CR). NK cell alloreactivity not only had a positive impact on the GVL effect, but also on the TRM through its beneficial effect on engraftment and aGVHD. NK cell GVH alloreactivity was associated with better survival even in AML patients who were in relapse at transplantation. OS was 30% versus 6% with no difference in RR, 32% versus 37%, respectively. In ALL, transplanted in relapse, the OS was 5% and there was no effect of NK alloreactivity. Currently, the Perugia group recommends proceeding to transplant for relapsed patients only in case of AML and when an NK alloreactive donor is available.
In extensive TCD transplantation, the expansion of the few T cells in the graft is delayed by the paucity of the starting population and not by immune suppression; immune recovery can take more than one year. The delayed immune recovery is mainly responsible for the high TRM (36%) due to infections. A significant drawback to the “mega dose” concept is that the harvest of CD34+ stem cells places a considerable demand not only on the donor but also on the apheresis unit and, additionally, is costly.
2.1.2. T cell replete graft
The Johns Hopkins and Seattle groups have evaluated the safety and efficacy of a RIC conditioning regimen based on FLU, cyclophosphamide (CY) and TBI plus transplantation of T replete BM and high dose (50mg/kg) intravenous post transplant CY on day +3 and +4 with tacrolimus and mycophenolate mofetil (MMF) as aGVHD prophylaxis. Graft failure occurred in 13% and grade II-IV aGVHD in 34% of patients receiving two doses of post transplant CY and 78% for patients receiving one dose. The non relapse mortality (NRM) at one year was 15% and the RR was 51%. The DFS at 6 months was 36% in the group of patients with advanced hematological malignancies. They also recently reported that when analyzing the same approach with different degrees of mismatch, they found no significant association between the number of HLA mismatches and the incidence of grade II-IV aGVHD or the
EFS48-52. Rizzieri et al. reported their experience using alemtuzumab (Campath) for in vivo host and donor TCD. The conditioning regimen was non-myeloablative (FLU and CY); post transplant GVHD prophylaxis included Campath with MMF ± CSP. The CD34+ stem cell dose was 13.5x106/kg. The TRM was low (10.2%) and grade II-IV aGVHD incidence was 16%.
However, the RR was 49% and the one year OS was 31%. More than half of the patients were not in first CR at transplantation. The standard-risk subgroup had a 63% OS at one year53;54.
Ogawa et al. proposed an ATG-based RIC protocol in the haploidentical setting for patients with advanced stage hematological malignancies. CSP and steroids were used as GVHD prophylaxis. Grade II-IV aGVHD was seen in 25% of patients, TRM was 15%, the RR was 23% and the DFS at 3 years was 55% 55. Recently Ciurca et al. reported a 42% DFS for poor risk patients after a RIC haploidentical transplant based on FLU, melphalan, thiotepa and ATG.
They also suggested that donor-specific anti-HLA antibodies are associated with a high rate of graft rejection in patients undergoing RIC haploidentical stem cell transplantation56;57. These approaches resulted in a high RR, probably related to the poor prognosis of the patients selected, and an expected higher rate of aGVHD, but a low TRM for patients older than usual and heavily pretreated. XJ Huang reported results from Beijing58;59 for an intensive in vivo immunosuppressive protocol without in vitro TCD. Patients were conditioned with a modified busulfan (BU) plus CY regimen including ATG and they received a combined G-CSF-primed BM and PBSC graft. CSP, MMF and methotrexate were given as GVHD prophylaxis. The G- CSF primed T cell dose was more than 100x106/kg; the incidence of grade II-IV aGVHD was 47% and extensive cGVHD was 22.6%. The two year probability of relapse was low, 12% for standard risk and 39% for high risk. TRM was 12% in standard risk and 20% in high risk. The two year DFS was 68% for standard risk, 42% for high risk and 22% for advanced disease. For AML the two year DFS was 78%, 57% and 22%, respectively, and for ALL, 74%, 35% and 14%, respectively. These results suggest that this approach can achieve an outcome comparable to the use of an HLA-matched sibling donor.
2.1.3. Conclusions on clinical results in haploidentical transplantation
Using the results cited above, it is evident that haploidentical transplant compares favorably with transplants from stem cell sources other than the HLA-matched sibling40 and, for some subgroups, the results can even compare favorably with matched sibling transplant. Further
improvements can be expected from the implementation of new strategies aimed at accelerating and strengthening post transplant immune reconstitution54, and, more specifically, from reinforcing anti-leukemia and anti-pathogen immunity. The positive results achieved in patients transplanted in CR1 and CR2 need to be transmitted to the hematologist community in order for them to refer patients for haploidentical transplant in the early stage of disease60. Moreover, when we now compare MUD transplants to haploidentical transplants, we have to keep in mind that MUD HSCT results only apply to patients who undergo transplantation, not considering those who do not find a MUD at all or relapse during the search. We also have to remember that for some poor prognosis patients with advanced or refractory AML haploidentical transplant may be the only chance of cure. For those patients, more must be done to optimize not only the GVL effect but also the conditioning regimen and the pre- transplant chemotherapy in order to improve their current very low LFS (Figure 3).
2.2. Principles of conditioning in haploidentical transplantation
2.2.1. Myeloablative strategies
Prior to allogeneic transplantation for AML, the conditioning regimen administered must be sufficiently immunosuppressive to ensure engraftment while contributing to the anti-leukemic impact of the procedure with no unacceptable toxicity. Currently a clear consensus is lacking about the superiority of any single ablative transplant regimen for AML in CR1. The classical myeloablative regimen for haploidentical transplant is based on TBI and CY with the addition of one or two agents like cytarabine (Ara-C), thiotepa and etoposide (VP-16) to increase immunosuppression and myeloablation. The Perugia group reported that enhancing immunosuppression and myeloablation by adding different agents such as ATG61, Ara-C and thiotepa to the standard conditioning based on CY and TBI did not ensure engraftment of TCD mismatched BM. Only the introduction of megadoses of stem cells was able to significantly improve the engraftment rate and the TRM. The new graft engineering technology permits the use of mobilized PBSC alone without the need of a BM harvest to achieve the target figures for the graft in terms of CD34+ stem cells and CD3+ T lymphocytes. Whether TBI was part of the conditioning regimen or not, did not seem to influence the engraftment when megadoses of CD34+ stem cells were given; therefore, TBI could be replaced by melphalan for patients who could not have TBI. Of the alkylating agents, CY has the most immunosuppressive activity but
FLU has emerged as a central agent to establish engraftment with less toxicity62. Consequently, FLU, as the immunosuppressive component of the conditioning, has been successfully combined using extensive variations in dose and type of partner agents. A study from O'Donnell et al. also suggests that FLU, BU and ATG could be superior to the classical BU- CY-ATG in allotransplant for AML. Thiotepa has excellent anti-leukemic and immunosuppressive activity and could also be a useful drug in the conditioning regimen for patients with advanced hematologic neoplasms. Moreover, a TBI thiotepa based conditioning regimen without post transplant immunosuppression also seems to be associated with a low incidence of leukemia relapse whether the TCD graft comes from a matched sibling or mismatched relative. In the matched sibling setting, the Perugia group observed a 0.12 and 0.28 probability of relapse, respectively, in patients with AML and ALL in CR1 and CR263;64. ATG also plays a central role in the conditioning regimen for haploidentical transplants because it persists in the plasma for several weeks62;65 and can thus deplete T cells not only from the recipient but also from the graft in the early post transplant period.
In their non-TCD strategy, the Beijing group chose a conditioning regimen composed of BU, Ara-C, CY, melphalan, lomustine (CCNU) and ATG. The major difference is the need for post transplant immunosuppression combining CSP, MMF and methotrexate. They also combined BM with PBSC to obtain higher numbers of CD34+ stem cells with fewer T cells59.
2.2.2. Reduced intensity conditioning in haploidentical transplantation
Even in haploidentical transplant, RIC approaches have resulted in decreased TRM and extended the option of HSCT to an older population with more comorbidities. The Tubingen group reported a RIC strategy based on partial TCD to ensure engraftment and with an intermediate intensity conditioning regimen based on melphalan, thiotepa, FLU and ATG or Muromonab-²CD3 (trade name Orthoclone, OKT3) with no other post transplant GVHD prophylaxis. The development of RIC strategies in allogeneic transplantation have showed that FLU and ATG are sufficiently immunosuppressive to allow engraftment. Nevertheless, these strategies, using reduced pre-transplant immunosuppression, require the use of more immunosuppression after the transplant to ensure engraftment. When a non-TCD strategy is used, the conditioning regimen can be less intensive, such as FLU, CY and TBI (2 Gy) or FLU, CY and Campath but it always requires post transplant GVHD prophylaxis using
compounds such as tacrolimus, MMF and CY as shown by the Johns Hopkins University group. These strategies are also usually based on the use of BM cells combined with PBSC to obtain more CD34+ stem cells with fewer T cells. This approach can achieve outcomes comparable with the use of an HLA-matched sibling donor. There is often a high aGVHD rate but a low RR and an acceptable TRM leading overall to good DFS.
2.3. Graft engineering in ex vivo T cell depleted haploidentical transplantation
In haploidentical transplant, proper graft preparation is crucial. The perfect graft should engraft quickly, prevent GVHD as well as post transplant infections and B cell lymphoproliferative disease while retaining the GVL effect to prevent relapse. While a single graft with all these characteristics is not yet a reality, managing each of these features independently is becoming increasingly feasible.
2.3.1. Positive CD34+ stem cell selection
The major advance in stem cell purification was the identification of the CD34 antigen and the development of the CD34 monoclonal antibody (mAb). The CD34 antigen was the first surface marker to identify a morphologically and immunologically heterogeneous hematopoietic immature cell population containing true stem cells that were able to reconstitute the entire hematopoiesis66;67. The CD34+ stem cell immunomagnetic positive selection, performed on the CliniMACS device (Miltenyi Biotec, Bergisch Gladdbach, Germany), permits isolation of megadoses of stem cells with very low numbers of T cells. In a subsequent Perugia trial68, the donor received 12µg/kg G-CSF subcutaneously twice daily to mobilize PBSC. After 72 hours of stimulation, the PBSC collection using a continuous flow cell separator (COBE Spectra) was performed. It is worth noting that the donors underwent a median of four leukaphereses. The peak of CD34+ mobilized stem cells in peripheral blood usually occurred on day 5, post initial stimulation. Sometimes two rounds of stimulation were necessary to reach the target of 10x106 CD34+ stem cells/kg for the recipient; a donor can only be successfully restimulated after a two weeks resting period. The apheresis target could be achieved in 90.2% of the donors.
Nevertheless, when three or four days of leukaphereses were needed, the CD34 selection required two separate selection procedures. The two selections are needed because a single
column can only be loaded with a maximum of 10x1010 total mononucleated cells (< 6x1010 according to the manufacturer) or 12.5x108 CD34+ stem cells (< 6x108 according to the manufacturer)69 with a mean recovery of 68% and a purity of 98%. In addition, the leukapheresis products should not be kept more than 24 hours before processing. In the Perugia experience, the median percentage CD34+ stem cells in the apheresis product was 0.72% (range, 0.11-2.66%) and the immunomagnetic selection provided a graft with an average purity of 93% CD34+ stem cells (range, 54.4-99.6%). The median viability of the selected stem cells was 97% (range, 95-100%) when evaluated with Trypan blue exclusion.
The median percentage for annexin V positive cells after selection was 21.1% (range, 8.7- 39%). The median recovery of CD34+ stem cells was 81% (range, 32-139%) with a 4.8 log TCD and 3.3 log B-cell depletion. The Perugia group reported that the targeted CD34+ stem cell dose could be obtained in 73.9% of the cases after a positive selection. The target number of CD3+ T cells (≤ 1x104/kg) was achieved in only 53.2% of the selections, while 45%
contained between 1x104 and 3x104 CD3+ T lymphocytes/kg and only 1.8% had more than 3x104/kg. There was also a published report that deeper platelet depletion by adjunctive low speed centrifugation could improve the stem cell recovery with no impact on the lymphocyte depletion (81% versus 71%)70. The megadose CD34 positive selection approach is very demanding, both for the donor and the technical staff, but allows successful engraftment without major GVHD. The major remaining problem is the slow immune reconstitution that increases the risk of death due to viral and fungal infections and compromises the early T lymphocyte mediated GVL effect.
2.3.2. Graft CD3/CD19+ lymphocyte depletion
The graft CD3/CD19 immunomagnetic depletion using the CliniMACS device retains more NK cells, monocytes and APC but the median T cell number in the graft is about 5 times higher than after a CD34 stem cell positive selection with a median CD34+ stem cell number of 5.2x106/kg. This approach has been used with RIC for which a higher T cell dose is needed for engraftment. The higher T cell dose resulted in a satisfactory engraftment rate with classical doses (around 5x106 CD34+ stem cell/Kg) of stem cells. The disadvantage was a higher rate of GVHD. The impact on DFS was difficult to evaluate because the median age of patients was high and most of them were in advanced stages of their disease. However, the results were
encouraging45. CD19 depletion was performed to decrease the risk of post transplant Epstein- Barr virus (EBV)-related lymphoproliferative disorders (LPD). It should be mentioned that, regardless of the selection approach, the balance between B and T cells is disturbed when one uses ATG in the conditioning regimen. This can increase the risk of EBV-LPD71.
3. Immune reconstitution after allogeneic hematopoietic stem cell transplantation
Successful reconstitution of cellular immunity following allogeneic HSCT reduces the risk of relapse and confers protection against opportunistic infection. The establishment of the donor immune system in the recipient takes months to years to complete. Consequently, it is crucial to analyze how the immune system recovers after transplant in order to be able to plan manipulations that could enhance the GVL effect without increasing the deleterious GVHD (Figure 4). 3.4.1. Immune reconstitution depends on de novo lymphoid generation and the function of mature cells contained in the graft. Post transplant NK cells can reconstitute within the first 100 days; however, B and T cell populations remain diminished for several months and the de novo thymic-related T cell generation can be definitively impaired in patients above 60 years old72. Immune reconstitution is very difficult to study because it depends on multiple parameters like age, sex, conditioning regimen, donor-host genetic disparities, source of stem cells, initial pathology and post transplant events such as GVHD and its treatment and infections, particularly if viral73.
3.1. Early post transplant immune response and induction of tolerance to the host
Nearly half a century ago, Burnet proposed that the function of the immune system was to distinguish self from non-self74-76. In this system, the neonatal period was essential in inducing self-tolerance by deletion of self-reactive lymphocytes and there was minimal accommodation for inducing tolerance in adults. The self-non-self model began to encounter problems in the late 1980s when immunologists recognized that T cells depend on other cells (APC) to pick up and then present the antigens to which they may respond and that the T cell response depends on whether APC are sending activation signals to the T cells as well77-81. In 1989, Janeway et
al. proposed that the old immunological paradigm had reached the limits of its usefulness82;83. He and others argued that the innate immune system is the real gatekeeper that determines whether the immune system responds. He also argued that the innate immune system uses ancient pattern-recognition receptors to make these decisions (recognizing a pathogen by its unchanging characteristics). In the 90s the new theoretical approach of the “Danger Model”, together with increased recognition of the role played by Tregs in the last decade, has opened the door to a more dynamic and functional approach of tolerance84;85. In 1994, Matzinger went several steps further by laying out the idea that APC respond to "danger signals" - most notably from cells undergoing injury, stress or necrosis (as opposed to apoptosis)84. The alarm signals released by these cells let the immune system know that there is a problem requiring an immune response. She argued that the immune response, orchestrated by T cells, occurs not because of a neonatal definition of "self", as in the Burnet model, nor because of ancient definitions of pathogens, as in the Janeway model, but as a result of a dynamic and constantly updated response to danger as defined by cellular damage. The Danger Model is quite broad covering topics as diverse as transplantation, maternal/fetal immunity, autoimmunity, cancer treatments and vaccines, but, although it explains how an immune response is triggered and how it ends, it does not account for why the immune system responds in different ways to different situations. As yet, the Danger Model has not won universal acceptance. Some immunologists believe that the immune response is mainly fueled by innate evolutionarily- conserved "pattern recognition receptors" that recognize patterns expressed by pathogens, and they do not see cell death in the absence of pathogens as a primary driver of the immune response. Even so, these ideas do not explain how the immune system rejects transplants or tumors or induces autoimmune diseases. Seong and Matzinger suggest that the "patterns" that the immune system recognizes on bacteria are not very different from the alarm signals released by damaged cells86. They suggest that, because life evolved in water, the hydrophobic portion of molecules is normally hidden in the internal parts of molecules or other structures (like membranes) and that their sudden exposure is a sign that some injury or damage has occurred. They also suggest that these are the most ancient alarm signals, that they are recognized by evolutionarily ancient systems of repair and remodeling and that the modern immune system has built upon this ancient system. Thus, bacteria and other organisms may have very similar alarm systems. They describe these ancient signals as Danger-Associated Molecular Patterns (DAMP). In a following article, Matzinger makes a case for what she now
