Elisabetta Zino - Academia.edu (original) (raw)
Papers by Elisabetta Zino
Hematology and Bone Marrow Transplantation Unit, Department of Oncology, San Raffaele Scientific ... more Hematology and Bone Marrow Transplantation Unit, Department of Oncology, San Raffaele Scientific Institute, Milano, Italy; Biostatistics Unit, Department of Health Sciences (DISSAL), University of Genova, Italy; Immunogenetics Laboratory, Unit of Immunohematology and Blood Transfusion, Division of Regenerative Medicine and Stem Cell Therapy, San Raffaele Scientific Institute, Milano, Italy; San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine and Stem Cell Therapy, San Raffaele Scientific Institute, Milano, Italy; Division of Hematology, Ospedale San Martino, Genova, Italy; Italian Bone Marrow Donor Registry, Ospedale Galliera, Genova, Italy; Division of Hematology and Bone Marrow Transplantation, University of Udine, Udine, Italy; Immunogenetics Laboratory, Ospedale San Martino, Genova, Italy; Tissue Typing Laboratory, Azienda Ospedaliera S. M. Misericordia, Udine, Italy; Institute of Hematology "L. & A. Seragnoli", University of...
Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di Te... more Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di Terlizzi, Anna Maria Parodi, Ruhena Sergeant, Miryam Martinetti, Andrea Bontadini, Francesca Bonifazi, Daniela Lisini, Benedetta Mazzi, Silvano Rossini, Paolo Servida, Fabio Ciceri, Chiara Bonini, Edoardo Lanino, Giuseppe Bandini, Franco Locatelli, Jane Apperley, Andrea Bacigalupo, Giovanni Battista Ferrara, Claudio Bordignon, and Katharina Fleischhauer
doi:10.1182/blood-2009-01-200378Prepublished online June 10, 2009;2009 114: 1437-1444€€€€Ematopoi... more doi:10.1182/blood-2009-01-200378Prepublished online June 10, 2009;2009 114: 1437-1444€€€€Ematopoietiche (CSE) e Terapia Cellulare, and the Italian Bone Marrow Donor RegistryFleischhauer and for the Gruppo Italiano Trapianto di Midollo Osseo, Cellule Staminale Garbarino, Valeria Miotti, Giuseppe Bandini, Alberto Bosi, Fabio Ciceri, Andrea Bacigalupo, KatharinaNicoletta Sacchi, Maria Pia Sormani, Jessica Marcon, Teresa Lamparelli, Renato Fanin, Lucia Roberto Crocchiolo, Elisabetta Zino, Luca Vago, Rosi Oneto, Barbara Bruno, Simona Pollichieni,€
doi:10.1182/blood-2003-04-1279Prepublished online October 23, 2003;€€€€Ferrara, Claudio Bordignon... more doi:10.1182/blood-2003-04-1279Prepublished online October 23, 2003;€€€€Ferrara, Claudio Bordignon and Katharina FleischhauerEdoardo Lanino, Giuseppe Bandini, Franco Locatelli, Jane Apperley, Andrea Bacigalupo, Giovanni B Bonifazi, Daniela Lisini, Benedetta Mazzi, Silvano Rossini, Paolo Servida, Fabio Ciceri, Chiara Bonini,Terlizzi, Anna Maria Parodi, Ruhena Sergeant, Miryam Martinetti, Andrea Bontadini, Francesca Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di€
Blood
Over 80% of stem cell transplantations (SCT) from unrelated donors (UD) are performed across alle... more Over 80% of stem cell transplantations (SCT) from unrelated donors (UD) are performed across allelic HLA-DPB1 disparities which have been associated with acute graft-versus-host disease (aGvHD) and, in 10/10 matched pairs, protection from disease relapse, while no significant correlation with overall survival (OS) could be revealed so far. We have previously developed an algorithm for non-permissive HLA-DP disparities involving an immunogenic T cell epitope (TCE) encoded by DPB1*0901, *1001 and *1701 (group 1), and, in a weaker form, by DPB1*0301, *1401 and *4501 (group 2) (Zino et al, Blood 2004). Here we report on the analysis of this algorithm in 627 UD SCT facilitated through the Italian Bone Marrow Donor Registry (IBMDR) and the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) between 1999 and 2006, performed for malignant disorders including acute myeloid or lymphoid leukaemia (n=320; 51%) and Hodgkin’s or non-Hodgkin’s lymphoma (n=86; 13.7%). 242 pairs (38.5%) were matched ...
Blood
Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunogl... more Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunoglobulin-like receptors (KIRs) in haploidentical hematopoietic stem cell transplantation (HSCT), has been associated with a selective graft versus leukemia (GvL) effect mediated by donor-derived alloreactive natural killer (NK) cells expressing KIRs whose ligands are missing in the recipient. In this study, we show that NK cells arising from hematopoietic stem cell progenitors after transplantation into haploidentical recipients, acquire a receptor repertoire that is compatible with patient-specific tolerance due to engagement of patient HLA ligands by inhibitory NK receptors. Using four-color immunofluorescence with monoclonal antibodies (mAbs) specific for the receptors CD94/NKG2A, KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2 and KIR3DL1, we have analyzed NK receptor reconstitution kinetics in eleven adult patients affected by acute myeloid (n=9) or lymphoblastic (n=2) leukemia, who underwent HSCT fr...
Blood
227 It is well established that the use of a donor matched for 9–10/10 alleles at HLA-A,-B,-C,-DR... more 227 It is well established that the use of a donor matched for 9–10/10 alleles at HLA-A,-B,-C,-DRB1,-DQB1 significantly improves overall survival (OS) after unrelated donor (UD) haematopoietic stem cell transplantation (HSCT). Whilst the matching status for HLA-DPB1 alleles has been shown to influence transplant complications (relapse and graft-versus-host disease (GVHD), its impact on survival has not been well defined. The current unmet need in clinical practice is an approach to stratify selection criteria when a clinician is confronted with the choice between several 10/10 or 9/10 matched unrelated donors. There is now considerable interest in exploring different types of matching criteria to define permissive HLA-DPB1 mismatches which may be associated with an improved outcome. We have previously shown that HLA-DPB1 permissiveness can be functionally defined by the characterization of shared T cell epitopes (TCE) recognized by alloreactive T cells. In this model, allelic HLA mi...
Blood
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem c... more Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on...
Blood
Alloreactive NK cells have been suggested to be important functional players in GvL activity afte... more Alloreactive NK cells have been suggested to be important functional players in GvL activity after haploidentical HSCT for high risk leukemia. In this study we have characterized NK cells differentiating from purified haploidentical CD34+ cells after transplantation into 16 patients who did (n=8) or did not (n=8) suffer acute leukemia relapse in a long term follow-up (median 208 days). The incidence of relapse in these patients was not correlated with the presence (n=9) or absence (n=7) of predicted donor NK alloreactivity (p=0.94). NK cells in the first month after transplantation were, regardless of the occurence of relapse, NKG2A+ (>95%) and KIR− (13%), thus resembling CD56bright NK cells from healthy donors. However, in contrast to mature CD56bright cells, the patients’ NK cells expressed heterogeneous intensities of CD56, were only partly positive for the lymph node homing markers CD62L and CCR7, and expressed a higher amount of Fcγ receptor III (CD16). Importantly, in contr...
Blood
1957 Background: In spite of the considerable progress achieved during the last decade in the fie... more 1957 Background: In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods: We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid ma...
Gut, 2015
ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, ... more ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides s...
Haematologica, 2010
CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune respon... more CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β ind...
Haematologica-the Hematology Journal, 2010
CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune respon... more CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem c... more Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors. Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search. Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.
Abstract 1957 Background: In spite of the considerable progress achieved during the last decade i... more Abstract 1957 Background: In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods: We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid malignancies (Acute Myeloid Leukemia (AML): 179; Myelodysplastic Syndrome (MDS): 27; Myeloproliferative Neoplasms: 17; Others: 7), received a fully myeloablative conditioning regimen, and infusion of donor T cells, either as part of the graft or as post-transplantation add-back. In addition to standard bone marrow morphological examination, post-transplantation follow-up comprised genomic HLA typing of bone marrow aspirate samples, aimed to identify HLA loss relapses. Results: A total of 83 relapses occurred, 72 and 11 after related and unrelated donor HSCT, respectively. We documented 21 relapses with genomic loss of the patient-specific mismatched HLA (25% of relapses). HLA loss occurred predominantly in patients with AML (n=19), but was also observed in patients with MDS (n=1) and primary myelofibrosis (n=1). None of the 11 cases of relapse after volunteer unrelated donor HSCT displayed HLA loss, whereas it was evident in 21 out of 72 (29%) relapses after family mismatched donor HSCT. Accordingly, the presence of multiple (more than 4) HLA mismatches between donor and recipient represented a significant risk factor for HLA loss (HR: 4.17, 95% CI: 0.88–19.82, p=0.07). None of the disease-related factors tested (morphological subtype, presence of dysplasia, hyperleukocytosis, cytogenetic and molecular profile) correlated significantly with HLA loss. In line with the hypothesis that selection of these leukemic variants may be elicited more frequently in the presence of a strong alloreactive immune pressure, HLA loss occurred more frequently in patients with clinical Graft-versu-Host Disease (GvHD), either acute (p=0.02) or chronic (p=0.007). Interestingly, HLA loss relapses occurred later than their “classical” counterparts (median time to relapse 307 vs 116 days, range 56–784 vs 10–579, p<0.0001), suggesting the existence of a long phase of equilibrium between leukemic cells and donor T cells before the occurrence of a de novo mutation and the outgrowth of immune escape variants. Of notice, HLA loss relapses shared several features with post-transplantation extramedullary relapses, suggesting a common biological drive leading to the two immune escape mechanisms. Conclusions: Loss of the mismatched HLA is a frequent mechanism of relapse for patients with high-risk myeloid malignancies, and is associated with donor-versus-host alloreactivity, as demonstrated by its significant correlation with the number of donor-recipient HLA mismatches and with the occurrence of GvHD. Ultimately, given the documented ineffectiveness of lymphocyte infusions from the original stem cell donor in patients with HLA loss relapses, efforts should be aimed towards the optimization of early detection tools and targeted therapeutic strategies specific for these peculiar and frequent relapse variants. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.
Little is known about the molecular char- acteristics of alloantigens recognized by alloreactive ... more Little is known about the molecular char- acteristics of alloantigens recognized by alloreactive T cells mediating hemato- logic stem cell graft rejection. In particu- lar, it has never been shown that such alloantigens can be encoded by HLA-DPb alleles. Indeed, matching for HLA-DP anti- gens is generally not considered to be of functional importance for the outcome of allogeneic bone
Biology of Blood and Marrow Transplantation, 2015
domestic inventory, respectively. Conclusion: CB extends transplant access to all patients & is e... more domestic inventory, respectively. Conclusion: CB extends transplant access to all patients & is especially important for minorities who are unlikely to have a matched URD. The fact that over 80% of units used in African & white Hispanic patients were domestic, that these patients will be very unlikely to identify a matched URD regardless of the size of the global volunteer donor registry, & that some minority patients do not have suitable CB grafts emphasizes the critical importance of the funding of US public CB banks.
Biology of Blood and Marrow Transplantation, 2015
A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction o... more A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction of permissive HLA mismatches, ie, those associated with lower clinical risks compared to their nonpermissive counterparts. For HLA-DPB1, a clinically prognostic model has been shown to be matching for T cell epitope (TCE) groups assigned by cross reactivity of T cells alloreactive to HLA-DPB1*09:01; however, the molecular basis of this observation is not fully understood. Here, we have mutated amino acids (aa) in 10 positions of HLA-DPB1*09:01 to other naturally occurring variants, expressed them by lentiviral vectors in B cell lines, and quantitatively measured allorecognition by 17 CD4 þ T cell effectors from 6 unrelated individuals. A significant impact on the median alloresponse was observed for peptide contact positions 9, 11, 35, 55, 69, 76, and 84, but not for positions 8, 56, and 57 pointing away from the groove. A score for the "functional distance" (FD) from HLA-DPB1*09:01 was defined as the sum of the median impact of polymorphic aa in a given HLA-DPB1 allele on T cell alloreactivity. Established TCE group assignment of 23 alleles correlated with FD scores of 0.5, 0.6 to 1.9 and !2 for TCE groups 1, 2, and 3, respectively. Based on this, prediction of TCE group assignment will be possible for any given HLA-DPB1 allele, including currently 367 alleles encoding distinct proteins for which T cell cross reactivity patterns are unknown. Experimental confirmation of the in silico TCE group classification was successfully performed for 7 of 7 of these alleles. Our findings have practical implications for the applicability of TCE group matching in unrelated HSCT and provide new insights into the molecular mechanisms underlying this model. The innovative concept of FD opens new potential avenues for risk prediction in unrelated HSCT.
Hematology and Bone Marrow Transplantation Unit, Department of Oncology, San Raffaele Scientific ... more Hematology and Bone Marrow Transplantation Unit, Department of Oncology, San Raffaele Scientific Institute, Milano, Italy; Biostatistics Unit, Department of Health Sciences (DISSAL), University of Genova, Italy; Immunogenetics Laboratory, Unit of Immunohematology and Blood Transfusion, Division of Regenerative Medicine and Stem Cell Therapy, San Raffaele Scientific Institute, Milano, Italy; San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine and Stem Cell Therapy, San Raffaele Scientific Institute, Milano, Italy; Division of Hematology, Ospedale San Martino, Genova, Italy; Italian Bone Marrow Donor Registry, Ospedale Galliera, Genova, Italy; Division of Hematology and Bone Marrow Transplantation, University of Udine, Udine, Italy; Immunogenetics Laboratory, Ospedale San Martino, Genova, Italy; Tissue Typing Laboratory, Azienda Ospedaliera S. M. Misericordia, Udine, Italy; Institute of Hematology "L. & A. Seragnoli", University of...
Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di Te... more Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di Terlizzi, Anna Maria Parodi, Ruhena Sergeant, Miryam Martinetti, Andrea Bontadini, Francesca Bonifazi, Daniela Lisini, Benedetta Mazzi, Silvano Rossini, Paolo Servida, Fabio Ciceri, Chiara Bonini, Edoardo Lanino, Giuseppe Bandini, Franco Locatelli, Jane Apperley, Andrea Bacigalupo, Giovanni Battista Ferrara, Claudio Bordignon, and Katharina Fleischhauer
doi:10.1182/blood-2009-01-200378Prepublished online June 10, 2009;2009 114: 1437-1444€€€€Ematopoi... more doi:10.1182/blood-2009-01-200378Prepublished online June 10, 2009;2009 114: 1437-1444€€€€Ematopoietiche (CSE) e Terapia Cellulare, and the Italian Bone Marrow Donor RegistryFleischhauer and for the Gruppo Italiano Trapianto di Midollo Osseo, Cellule Staminale Garbarino, Valeria Miotti, Giuseppe Bandini, Alberto Bosi, Fabio Ciceri, Andrea Bacigalupo, KatharinaNicoletta Sacchi, Maria Pia Sormani, Jessica Marcon, Teresa Lamparelli, Renato Fanin, Lucia Roberto Crocchiolo, Elisabetta Zino, Luca Vago, Rosi Oneto, Barbara Bruno, Simona Pollichieni,€
doi:10.1182/blood-2003-04-1279Prepublished online October 23, 2003;€€€€Ferrara, Claudio Bordignon... more doi:10.1182/blood-2003-04-1279Prepublished online October 23, 2003;€€€€Ferrara, Claudio Bordignon and Katharina FleischhauerEdoardo Lanino, Giuseppe Bandini, Franco Locatelli, Jane Apperley, Andrea Bacigalupo, Giovanni B Bonifazi, Daniela Lisini, Benedetta Mazzi, Silvano Rossini, Paolo Servida, Fabio Ciceri, Chiara Bonini,Terlizzi, Anna Maria Parodi, Ruhena Sergeant, Miryam Martinetti, Andrea Bontadini, Francesca Elisabetta Zino, Guido Frumento, Sarah Marktel, Maria Pia Sormani, Francesca Ficara, Simona Di€
Blood
Over 80% of stem cell transplantations (SCT) from unrelated donors (UD) are performed across alle... more Over 80% of stem cell transplantations (SCT) from unrelated donors (UD) are performed across allelic HLA-DPB1 disparities which have been associated with acute graft-versus-host disease (aGvHD) and, in 10/10 matched pairs, protection from disease relapse, while no significant correlation with overall survival (OS) could be revealed so far. We have previously developed an algorithm for non-permissive HLA-DP disparities involving an immunogenic T cell epitope (TCE) encoded by DPB1*0901, *1001 and *1701 (group 1), and, in a weaker form, by DPB1*0301, *1401 and *4501 (group 2) (Zino et al, Blood 2004). Here we report on the analysis of this algorithm in 627 UD SCT facilitated through the Italian Bone Marrow Donor Registry (IBMDR) and the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) between 1999 and 2006, performed for malignant disorders including acute myeloid or lymphoid leukaemia (n=320; 51%) and Hodgkin’s or non-Hodgkin’s lymphoma (n=86; 13.7%). 242 pairs (38.5%) were matched ...
Blood
Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunogl... more Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunoglobulin-like receptors (KIRs) in haploidentical hematopoietic stem cell transplantation (HSCT), has been associated with a selective graft versus leukemia (GvL) effect mediated by donor-derived alloreactive natural killer (NK) cells expressing KIRs whose ligands are missing in the recipient. In this study, we show that NK cells arising from hematopoietic stem cell progenitors after transplantation into haploidentical recipients, acquire a receptor repertoire that is compatible with patient-specific tolerance due to engagement of patient HLA ligands by inhibitory NK receptors. Using four-color immunofluorescence with monoclonal antibodies (mAbs) specific for the receptors CD94/NKG2A, KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2 and KIR3DL1, we have analyzed NK receptor reconstitution kinetics in eleven adult patients affected by acute myeloid (n=9) or lymphoblastic (n=2) leukemia, who underwent HSCT fr...
Blood
227 It is well established that the use of a donor matched for 9–10/10 alleles at HLA-A,-B,-C,-DR... more 227 It is well established that the use of a donor matched for 9–10/10 alleles at HLA-A,-B,-C,-DRB1,-DQB1 significantly improves overall survival (OS) after unrelated donor (UD) haematopoietic stem cell transplantation (HSCT). Whilst the matching status for HLA-DPB1 alleles has been shown to influence transplant complications (relapse and graft-versus-host disease (GVHD), its impact on survival has not been well defined. The current unmet need in clinical practice is an approach to stratify selection criteria when a clinician is confronted with the choice between several 10/10 or 9/10 matched unrelated donors. There is now considerable interest in exploring different types of matching criteria to define permissive HLA-DPB1 mismatches which may be associated with an improved outcome. We have previously shown that HLA-DPB1 permissiveness can be functionally defined by the characterization of shared T cell epitopes (TCE) recognized by alloreactive T cells. In this model, allelic HLA mi...
Blood
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem c... more Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on...
Blood
Alloreactive NK cells have been suggested to be important functional players in GvL activity afte... more Alloreactive NK cells have been suggested to be important functional players in GvL activity after haploidentical HSCT for high risk leukemia. In this study we have characterized NK cells differentiating from purified haploidentical CD34+ cells after transplantation into 16 patients who did (n=8) or did not (n=8) suffer acute leukemia relapse in a long term follow-up (median 208 days). The incidence of relapse in these patients was not correlated with the presence (n=9) or absence (n=7) of predicted donor NK alloreactivity (p=0.94). NK cells in the first month after transplantation were, regardless of the occurence of relapse, NKG2A+ (>95%) and KIR− (13%), thus resembling CD56bright NK cells from healthy donors. However, in contrast to mature CD56bright cells, the patients’ NK cells expressed heterogeneous intensities of CD56, were only partly positive for the lymph node homing markers CD62L and CCR7, and expressed a higher amount of Fcγ receptor III (CD16). Importantly, in contr...
Blood
1957 Background: In spite of the considerable progress achieved during the last decade in the fie... more 1957 Background: In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods: We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid ma...
Gut, 2015
ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, ... more ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides s...
Haematologica, 2010
CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune respon... more CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β ind...
Haematologica-the Hematology Journal, 2010
CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune respon... more CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem c... more Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors. Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search. Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.
Abstract 1957 Background: In spite of the considerable progress achieved during the last decade i... more Abstract 1957 Background: In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods: We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid malignancies (Acute Myeloid Leukemia (AML): 179; Myelodysplastic Syndrome (MDS): 27; Myeloproliferative Neoplasms: 17; Others: 7), received a fully myeloablative conditioning regimen, and infusion of donor T cells, either as part of the graft or as post-transplantation add-back. In addition to standard bone marrow morphological examination, post-transplantation follow-up comprised genomic HLA typing of bone marrow aspirate samples, aimed to identify HLA loss relapses. Results: A total of 83 relapses occurred, 72 and 11 after related and unrelated donor HSCT, respectively. We documented 21 relapses with genomic loss of the patient-specific mismatched HLA (25% of relapses). HLA loss occurred predominantly in patients with AML (n=19), but was also observed in patients with MDS (n=1) and primary myelofibrosis (n=1). None of the 11 cases of relapse after volunteer unrelated donor HSCT displayed HLA loss, whereas it was evident in 21 out of 72 (29%) relapses after family mismatched donor HSCT. Accordingly, the presence of multiple (more than 4) HLA mismatches between donor and recipient represented a significant risk factor for HLA loss (HR: 4.17, 95% CI: 0.88–19.82, p=0.07). None of the disease-related factors tested (morphological subtype, presence of dysplasia, hyperleukocytosis, cytogenetic and molecular profile) correlated significantly with HLA loss. In line with the hypothesis that selection of these leukemic variants may be elicited more frequently in the presence of a strong alloreactive immune pressure, HLA loss occurred more frequently in patients with clinical Graft-versu-Host Disease (GvHD), either acute (p=0.02) or chronic (p=0.007). Interestingly, HLA loss relapses occurred later than their “classical” counterparts (median time to relapse 307 vs 116 days, range 56–784 vs 10–579, p<0.0001), suggesting the existence of a long phase of equilibrium between leukemic cells and donor T cells before the occurrence of a de novo mutation and the outgrowth of immune escape variants. Of notice, HLA loss relapses shared several features with post-transplantation extramedullary relapses, suggesting a common biological drive leading to the two immune escape mechanisms. Conclusions: Loss of the mismatched HLA is a frequent mechanism of relapse for patients with high-risk myeloid malignancies, and is associated with donor-versus-host alloreactivity, as demonstrated by its significant correlation with the number of donor-recipient HLA mismatches and with the occurrence of GvHD. Ultimately, given the documented ineffectiveness of lymphocyte infusions from the original stem cell donor in patients with HLA loss relapses, efforts should be aimed towards the optimization of early detection tools and targeted therapeutic strategies specific for these peculiar and frequent relapse variants. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.
Little is known about the molecular char- acteristics of alloantigens recognized by alloreactive ... more Little is known about the molecular char- acteristics of alloantigens recognized by alloreactive T cells mediating hemato- logic stem cell graft rejection. In particu- lar, it has never been shown that such alloantigens can be encoded by HLA-DPb alleles. Indeed, matching for HLA-DP anti- gens is generally not considered to be of functional importance for the outcome of allogeneic bone
Biology of Blood and Marrow Transplantation, 2015
domestic inventory, respectively. Conclusion: CB extends transplant access to all patients & is e... more domestic inventory, respectively. Conclusion: CB extends transplant access to all patients & is especially important for minorities who are unlikely to have a matched URD. The fact that over 80% of units used in African & white Hispanic patients were domestic, that these patients will be very unlikely to identify a matched URD regardless of the size of the global volunteer donor registry, & that some minority patients do not have suitable CB grafts emphasizes the critical importance of the funding of US public CB banks.
Biology of Blood and Marrow Transplantation, 2015
A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction o... more A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction of permissive HLA mismatches, ie, those associated with lower clinical risks compared to their nonpermissive counterparts. For HLA-DPB1, a clinically prognostic model has been shown to be matching for T cell epitope (TCE) groups assigned by cross reactivity of T cells alloreactive to HLA-DPB1*09:01; however, the molecular basis of this observation is not fully understood. Here, we have mutated amino acids (aa) in 10 positions of HLA-DPB1*09:01 to other naturally occurring variants, expressed them by lentiviral vectors in B cell lines, and quantitatively measured allorecognition by 17 CD4 þ T cell effectors from 6 unrelated individuals. A significant impact on the median alloresponse was observed for peptide contact positions 9, 11, 35, 55, 69, 76, and 84, but not for positions 8, 56, and 57 pointing away from the groove. A score for the "functional distance" (FD) from HLA-DPB1*09:01 was defined as the sum of the median impact of polymorphic aa in a given HLA-DPB1 allele on T cell alloreactivity. Established TCE group assignment of 23 alleles correlated with FD scores of 0.5, 0.6 to 1.9 and !2 for TCE groups 1, 2, and 3, respectively. Based on this, prediction of TCE group assignment will be possible for any given HLA-DPB1 allele, including currently 367 alleles encoding distinct proteins for which T cell cross reactivity patterns are unknown. Experimental confirmation of the in silico TCE group classification was successfully performed for 7 of 7 of these alleles. Our findings have practical implications for the applicability of TCE group matching in unrelated HSCT and provide new insights into the molecular mechanisms underlying this model. The innovative concept of FD opens new potential avenues for risk prediction in unrelated HSCT.