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MLET 5404 1–3

Immunology Letters xxx (2013) xxx– xxx

Contents lists available at ScienceDirect

Immunology Letters

jo u r n al homep age: www.elsev ier .com/ locate / immlet

eview

egative depletion of �/�+ T cells and of CD19+ B lymphocytes: novel frontier to optimize the effect of innate immunity inLA-mismatched hematopoietic stem cell transplantation

ranco Locatelli a,b,∗, Aurelie Bauqueta, Giuseppe Palumboa,rancesca Morettaa, Alice Bertainaa

Department of Pediatric Hematology and Oncology, IRCCS Ospedale Bambino Gesù, Rome, ItalyUniversity of Pavia, Italy

r t i c l e i n f o

rticle history:vailable online xxx

eywords:llogeneic hematopoietic stem cell

ransplantationelective T-cell depletionK cell maturation�+ T cells

a b s t r a c t

In recent years, infusion of T-cell depleted hematopoietic stem cells from an HLA-haploidentical relativehas been shown to represent a suitable and effective, alternative option in patients in need of an allograftwho lack an HLA-identical relative. In particular, this type of allograft is associated with the enormousadvantage of offering an immediate transplant treatment to virtually all pediatric patients without anHLA-matched donor, whether related or unrelated, or a suitable umbilical cord blood unit. Several stud-ies have shown that in patients given a T-cell depleted transplant relevant part of the anti-leukemiaeffect is mediated by alloreactive (i.e. KIR/HLA mismatched) Natural Killer cells originated from donorhematopoietic stem cells. After infusion of positively selected hematopoietic stem cell, fully functioningNatural Killer cells emerge in the recipient peripheral blood, persisting over time, only several weeks

after the allograft. We have developed a new method of T-cell depletion (based on the physical elimina-tion of mature T cells carrying alpha and beta chains of the T-cell receptor), which permits to maintainmature donor-derived alloreactive Natural Killer cells and ��+ T cells in the graft. We, thus, started aformal study in children with hematological disorders aimed at evaluating the safety and efficacy of thisapproach. Preliminary results on 60 children transplanted so far after this type of graft manipulation areparticularly promising.

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. Introduction

Over the last 4 decades, allogeneic hematopoietic stem cellransplantation (allo-HSCT) from an HLA-matched donor, eitherelated or unrelated, has been increasingly used to treat patientsffected by several malignant or non-malignant hematological dis-rders [1]. However, only 25% of patients who need or could benefitrom an allograft have an HLA-identical sibling, and for less than

Please cite this article in press as: Locatelli F, et al. Negative depletitier to optimize the effect of innate immunity in HLA-mismatched

http://dx.doi.org/10.1016/j.imlet.2013.09.027

0% of the remaining patients a suitable, HLA-compatible, unre-ated volunteer can be found [2]. Moreover, the median time toocate a suitable unrelated donor is in the order of 3–4 months. In

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloideukemia; GvHD, graft-versus-host disease; GvL, graft-versus-leukemia; HSC,ematopoietic stem cell; HSCT, hematopoietic stem cell transplantation; MDS,yelodysplastic syndromes; NK, natural killer; TCR, T cell receptor; UCB, umbilical

ord blood.∗ Corresponding author at: IRCCS Bambino Gesù Children’s Hospital, Piazza

ant’Onofrio 4, 00165 Rome, Italy. Tel.: +39 06 68592678; fax: +39 06 68592292.E-mail address: franco.locatelli@opbg.net (F. Locatelli).

165-2478/$ – see front matter © 2013 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.imlet.2013.09.027

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© 2013 Published by Elsevier B.V.

the absence of an HLA-matched donor, alternative donor/sources ofhematopoietic stem cells (HSC), such as unrelated umbilical cordblood (UCB) and HLA-haploidentical relatives, are being increas-ingly used [3,4]. In particular, the majority of patients have afamily member, identical for one HLA haplotype and fully mis-matched for the other (i.e. haploidentical), who could immediatelyserve as HSC donor [5]. It is therefore evident that HSCT froman HLA-haploidentical relative (haplo-HSCT) offers an immediatetransplant treatment for those patients who dot have a matcheddonor or a suitable UCB unit available.

For many years, relevant obstacles to a wide use ofHLA-haploidentical family donors have been represented by graft-versus-host disease (GvHD) and graft rejection, mediated by donorand host alloreactive T-cell response, respectively. In particular, inthis context of great immune genetic disparity between patient anddonor, although mature donor T cells present in the graft facili-tate T-cell reconstitution, they are responsible for the occurrence of

on of �/�+ T cells and of CD19+ B lymphocytes: A novel fron-hematopoietic stem cell transplantation. Immunol Lett (2013),

GvHD, a severe, sometimes fatal, complication, which also impairspatient’s immune reconstitution [6]. Strategies to prevent GvHDafter HLA-haploidentical allo-HSCT based on either pharmacolog-ical immunosupression or T-cell depletion of the graft have been

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eveloped over the time. In particular, a milestone in the history ofaplo-HSCT was the demonstration that an efficient T-cell deple-ion of the graft is able to prevent both acute and chronic GvHDven when using a related donor differing at the three major HLAoci [7]. The other major breakthrough in the field of haplo-HSCT

as the use of “megadoses” of G-CSF-mobilized peripheral bloodonor HSC. Indeed, the infusion of huge numbers of hematopoieticrogenitors has been shown in the experimental model to be able tovercome the barrier of HLA incompatibility in the donor/recipientair and to elude the residual anti-donor cytotoxic T-lymphocytectivity of the recipient [7].

. Clinical results of T-cell depleted HLA-haploidenticalllogeneic HSCT

An effective clinical translation of the infusion of “megadoses”f G-CSF-mobilized peripheral blood donor HSC was first reportedn adults with acute leukemia by the group of Perugia, whichliminated donor T cells through soybean lectin agglutination and-rosetting with sheep erythrocytes [8]. The infusion of thesemegadoses” of T-cell-depleted HSC without any subsequent phar-acological GvHD prophylaxis, resulted into an engraftment rate

reater than 90% and a cumulative incidence of both grades II–IVcute and chronic GvHD below 10% [8]. Subsequently, the sameroup has adopted a more standardized method of T-cell deple-ion based on the positive selection of CD34 cells from peripherallood by magnetic beads using the CliniMACS® (Miltenyi) sys-em. The clinical trials performed using this approach of T-cellepletion have confirmed that sustained engraftment of donorematopoiesis, without the occurrence of GvHD, can be obtained inhe majority of adult patients and that a substantial proportion ofhem, especially those affected by acute myeloid leukemia (AML)r myelodysplastic syndromes (MDS), became long-term survivors9,10]. The feasibility and efficacy of haplo-HSCT was reproducedlso in children with acute leukemia, as documented in few pilottudies enrolling a limited number of patients [11,12]. A largertudy performed by The Acute Leukemia and Pediatric Workingarties of the European Blood and Marrow Transplantation (EBMT)roup analyzed the outcome of a cohort of children with acute

ymphoblastic leukemia (ALL) given T-cell depleted haplo-HSCT.his study showed that the 5-year leukemia-free survival (LFS) forhildren transplanted in complete remission was about 30%, thisnding indicating that haplo-HSCT is a useful treatment for patients

n morphological remission of this disease. Furthermore, this studyhowed that the stem cell dose infused in haplo-HSCT is crucial, asatients receiving a dose of CD34+ cells greater than 12x106 pro-enitors/kg had a better clinical outcome in comparison to thoseransplanted with a lower number of cells [13].

.1. Anti-leukemia effect of T-cell depleted HLA-haploidenticalllogeneic HSCT

The efficacy of allogeneic HSCT in patients with malignanciess mainly due to the so-called “graft-versus-tumor” (GVT) effect,

hich, in unmanipulated transplants, is largely mediated by donor-erived T cells present in the graft and recognizing allogeneiceptide-human leukocyte antigen (HLA)-complexes displayed byalignant cells [14]. In view of the role played by donor T cells inediating the GvL effect, it was anticipated that a relevant pro-

ortion of patients with acute leukemia given this type of allograftould have experienced disease relapse. This expectation was only

Please cite this article in press as: Locatelli F, et al. Negative depletitier to optimize the effect of innate immunity in HLA-mismatched

http://dx.doi.org/10.1016/j.imlet.2013.09.027

artly confirmed by the clinical results, as several studies havehown that protection against disease relapse after T-cell depletedLA-haploidentical allo-HSCT in adult patients with AML and chil-ren with ALL occurs in the presence of particular HLA-allele

PRESSetters xxx (2013) xxx– xxx

combinations between donor and recipient, a phenomenon knownas alloreactivity mediated by natural killer (NK) cells [15–17]. Thisreaction offers the advantage of inducing GvL without promotingGvHD occurrence, because non-hematopoietic tissues of the recip-ient are protected from donor NK cell-dependent alloreactivity, asthey lack ligands for the activating receptors of NK cells [16]. Indeed,the ligands recognized by activating NK receptors are expressedor upregulated in tissues of different histotypes only upon cellstress, tumor transformation or viral infection; this may providethe molecular basis to explain the inability of NK cells to attacknormal tissues (with the exception of the hematopoietic cells thatconstitutively express such ligands). In humans, according to theconcept of “missing-self recognition”, donor NK cell alloreactivitycan be predicted to occur in approximately 50% of patients givenan HLA-disparate family donor allo-HSCT.

The donor NK-mediated GvL effect was particularly evidentwhen patients with acute leukemia were transplanted in morpho-logical complete remission and, in children and young adults, whenthe donor was the mother [10,12,16,17].

In a seminal study performed to investigate the kinetics ofNK cell reconstitution, it was shown that, after transplantation ofCD34+-selected cells, mature, fully functioning NK cells, derivedfrom the differentiation of HSC, emerge in the recipient periph-eral blood only several weeks after the allograft, while in the earlypost-transplant period immature, poorly functioning KIR−NKG2A+

NK cells predominate [17]. This observation underlined the con-cept that patients transplanted with T-cell depleted HSC from anNK-alloreactive relative cannot benefit from the NK-mediated GvLeffect in the early post-transplant period and provides the ratio-nale either for strategies of adoptive infusion of ex vivo-activatedNK cells [18] during this time-window or for alternative approachesof graft manipulation, such as that based on the selective physicalremoval of ��+ T lymphocytes [19,20]. In fact, T lymphocytes car-rying the �/�chains of the T-cell receptor TCR are the lymphocytesubset responsible for the occurrence of GvHD, and, thus, their elim-ination allows preventing the occurrence of this life-threateningcomplication [6]. The selective physical removal of ��+ T lym-phocyte makes it possible to provide functional �� T cells andalloreactive NK cells that may exert their anti-leukemia activitysoon after transplantation, thus lowering the risk of rapid leukemiarelapses occurring in cases of partial responses to the conditioningregimen and/or in rapidly proliferating leukemias. The subsequentgeneration of alloreactive NK cells from grafted HSC assures a lateand more durable protection.

Mature NK cells can display not only anti-leukemia activity butalso important anti-viral function that could be beneficial to HSCTrecipients. Moreover, they also contribute to prevent GvHD throughthe killing of recipient dendritic cells [6,21].

�� T cells (also termed “innate-like” T cells or “transitional”T cells) belong to the adaptive arm of the immune system [22].These cells are capable of recognizing their targets in an MHC-independent manner through activating receptors (among others,��-TCR, NKG2D, TLRs, DNAM-1) and display a pre-activated phe-notype which allows rapid cytokine production (IFN-�, TNF-�) andstrong cytotoxic response upon activation. The fact that �� T cells,in contrast to �� T cells, are not restricted by MHC molecules makesthem unlikely to elicit GvHD, based on HLA alloreactivity. A num-ber of preclinical and clinical observations point to their potentiallybeneficial role in cancer medicine. In fact, �� T-cell functions areheterogeneous, ranging from protection against intra- and extra-cellular pathogens, to tumor, immune response modulation andmaintenance of tissue homeostasis [23–26]. Notably, similar to

on of �/�+ T cells and of CD19+ B lymphocytes: A novel fron-hematopoietic stem cell transplantation. Immunol Lett (2013),

NK cells, both the spontaneous and cytokine induced anti-tumoractivities of �� T cells appear more prominent against hematolog-ical cancers than other types of malignancies [27]. Human �� Tcells, which account for 1–10% of peripheral blood lymphocytes,

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re polyclonal but frequently express TCRs composed of V�9 and�2 chains. By contrast, the majority of tissue-associated human� T cells express V�1+ TCRs, which are also found in about 5–30%f circulating �� T cells. A better recovery of �� T lymphocytes afternmanipulated allogeneic bone marrow transplantation has beenssociated with an increased probability of leukemia-free survival,ithout any augmented risk of GvHD [28].

As mentioned above, we recently tested a new method of graftanipulation in HLA-haploidentical HSCT recipients. This method

elies on the negative selection of T lymphocytes carrying the � and chains of TCR and of B lymphocytes. We started the program of T-ell depleted haplo-HSCT using the negative selection of �� TCR Tells in November 2010. The study is officially registered at the clini-altrials.gov website with the following number: NCT01810120. Soar, we have transplanted with the method of negative depletion of� TCR T cells 60 patients with either hematological malignanciesr with non-malignant disorders. The data collected to date clearlyemonstrates that this method of graft manipulation is reliable andeproducible, leading to around a 4.1-log and 3.1-log depletion of� TCR T cells and B cells, respectively. The residual T-cell con-

ent was below the safe threshold (i.e. 1 × 105/kg recipient bodyeight) for performing HLA-haploidentical HSCT without any post-

ransplant immune suppression in all donor depletions performed.he preliminary clinical results of the new method of T-cell deple-ion are promising, since patients receiving an �� T lymphocyte/Bymphocyte-depleted allograft have a negligible risk of developingoth severe acute GvHD and chronic GvHD, while being protectedrom life-threatening infections [29]. Interestingly, the early T-cellecovery is mainly contributed to by �� T lymphocytes, which rep-esent the predominating T-cell subset till 45–60 days after thellograft (Locatelli F, personal unpublished data, 2013).

From all these data, it is therefore evident that major advancesn the transplant therapy of high risk leukemias have been pos-ible thanks to the discovery and the functional characterizationf inhibitory and activating NK receptors that are expressed onoth NK cells and �� T cells and to the development of novelpproaches of graft manipulation. These insights have really theotential capacity to dramatically modify in the near future theeld of allogeneic HSCT and to change the fate of patients withany life-threatening disorders.

cknowledgments

This work was partially supported by grants from AIRC (Asso-iazione Italiana Ricerca sul Cancro, progetto speciale 5xmilleINNATE IMMUNITY IN CANCER. MOLECULAR TARGETING ANDELLULAR THERAPY”), CNR (Consiglio Nazionale delle Ricerche),IUR (Ministero dell’Istruzione, Università e della Ricerca, Progetto

ilevante Interesse Nazionale 2010), Ministero della salute (Pro-etto Finalizzato 2011) and IRCCS Ospedale Pediatrico Bambinoesù to F.L.

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