ALBERT

Description: 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.

Description: 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 contrast to mature CD56bright cells, which constitrutively express the high affinity αβγ IL-2 receptor, thus releasing γ-IFN in response to low dose IL2, the patients’ NK cells lacked IL-R α (CD25) and did not release cytokines in response to low-dose IL2, nor, most importantly, when challenged with leukemic blasts. γ-IFN release induced by leukemic blasts could be restored by inhibition of NKG2A while cytotoxicity, which was consistently lower as compared to that of mature CD56+ cells, could not. Our data suggest that NK cells differentiating in patients from CD34+ progenitors after haploidentical HSCT have important phenotipical and functional differences from both subsets of mature NK cells, accounting for an impaired in vivo GvL potential.

Description: Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) is a promising therapeutic option for patients lacking a fully compatible donor. Due to extensive T cell depletion, Natural Killer (NK) cell activity represents the only immunological protection against disease relapse for the first months after haplo-HSCT. Clinical studies have associated donor-recipient incompatibility for Human Leukocyte Antigen (HLA) ligands of Killer Immunoglobulin-like Receptors (KIR), with a marked anti-leukemic activity. Alloreactive donor NK cells carrying a single KIR whose ligand is missing in the recipient mediate a potent graft vs. leukemia effect, resulting in reduced incidence of relapse and increased Overall Survival (OS). These exciting results have recently been challenged by conflicting clinical and biological data from different groups. In the present study, we have characterized reconstitution of NK cells, in particular of alloreactive single-KIR+ NK cells, in 58 patients who received CD34+ selected haplo-HSCT for high-risk hematologic malignancies. One month after haplo-HSCT CD56bright/CD56dim NK cell subsets were subverted in their proportions and phenotypic features, accounting for enrichment in maturation intermediates. We show that CD25 and CD117 deregulation by CD56bright, and NKG2A and CD62L by CD56dim, are intrinsic to NK cell physiologic differentiation and support a sequential CD56bright-to-CD56dim NK cell maturation. Consistently, the in vitro functional potential of these maturation intermediates against leukemic blasts was heavily impaired, both in terms of cytotoxicity and of cytokine release. Full mature receptor repertoire reconstitution took at least three months. Alloreactive single-KIR+ NK cells had highly variable frequency ranging from less than 1% to more than 30% of NK cells circulating at 90–120 days after transplantation, independently from predicted NK alloreactivity. Importantly, out of three patients with predicted NK alloreactivity, none had a relative expansion of alloreactive single-KIR+ cells, accounting for less than 1% of circulating NK cells in two of them. As demonstrated by flow cytometric analysis of NK cell CD107a mobilization in response to the HLA class I negative target 721.221, single-KIR+ NK cells at three months after haplo-HSCT showed a not yet fully developed functional reactivity, which was recovered to donor-levels only at later time-points. In line with these observations, clinical outcome of haplo-HSCT was not affected in any way by the presence of donor NK alloreactivity. The incidence of relapse was virtually identical in patients transplanted from alloreactive or non-alloreactive donors. Taken together, our data shed new light onto the kinetics of NK cell differentiation in vivo and suggest that NK alloreactivity could be best exploited by the use of mature donor single-KIR+ selected alloreactive NK cells.

Description: Abstract 4494 1.Background Mismatches of minor Histocompatibility antigens (mHAg) have been considered as an important immunogenetic factor influencing outcomes and immune responses following allogeneic stem-cell transplantation (alloSCT) despite fully matched HLA of donor and recipient. 2. Aim Aim of this study was to assess whether mHAg incompatibilities may affect overall survival (OS), progression free survival (PFS) and GVHD incidence (acute and chronic, aGVHD and cGVHD) in patients receiving alloSCT for lymphoid malignancies. 3. Methods Sixty-four consecutive patients with B-cell lymphomas who underwent alloSCT were studied. Ten patients had chronic lymphocytic leukemia (CLL, 15.8%), 3 had follicular lymphoma (FCL, 4.7%), 1 had diffuse large B cell lymphoma (DLBCL, 1,5%), 17 had Hodgkin's lymphoma (26.5%) and 33 had multiple myeloma (51.5%). All underwent peripheral blood stem-cells allograft with non-myeloablative (13 patients, 20%) or reduced intensity (51 pts, 80%) Fludarabine-based conditioning; GVHD prophylaxis included methotrexate and oral cyclosporine +/- micomofetil fenolate in case of matched unrelated donors. Median age was 51 years (range 18-66); 35 patients were male (55%), median number of previous chemotherapies was 3 (0-7), 49 patients had a previous autologous transplant (76%). Twenty-five patients were in complete remission (CR, 39%), 30 and 9 were in partial response and progression (PR 47% and PD 14%). Karnofsky performance status (PS) was 〉80% in 50 patients (78%). Forty-four patients allografted from HLA-matched siblings (69%), 20 from matched unrelated donor (31%): all were matched at allelic level for HLA-A, -B, -Cw, -DRB1 and -DQB1 loci. Allelic mHAs typing was performed by PCR with sequence-specific primers for 14 autosomic mHAg and H-Y. Host versus Graft or Graft versus Host direction of immune responses in donor/recipient pairs was analyzed with use of the minor Histocompatibility Database of Leiden University Medical Center. OS and PFS were analyzed with Kaplan-Meier method and log-rank test. aGVHD and cGVHD were analyzed with a multivariate logistic regression including as covariates age, sex, previous lines of chemotherapy, disease, pre-transplant status, PS and mHAs mismatches; grade 〉=2 aGVHD and extensive cGVHD were considered events. 4. Results Median follow-up was 34 months (2-83). One-year OS was 85%, 2- and 3-years OS were 82% and 77%. One-year PFS was 61%, 2- and 3-years PFS were 49% and 41%. The univariate analysis which considered transplant characteristics showed that OS and PFS were significantly affected by disease status at transplant (p

Description: Purpose: Haploidentical hematopoietic stem cell transplantation (haplo-SCT) provides an option for cancer patients lacking a compatible donor. However, the delayed immune reconstitution increases incidence of opportunistic infections and disease relapse. We conducted a phase I-II prospective trial to evaluate the effect of escalating doses of CD8-depleted (Miltenyi Device) donor lymphocyte infusions (DLIs) following RIC haplo-SCT. Here, we present the data on the immune reconstitution. Patients and Methods: Twenty-eight patients (pts) with advanced lymphoproliferative diseases (n=24) or acute myeloid leukaemia (n=4) were enrolled in this study. Fifteen (54%) of 28 pts had refractory disease. All pts received a thiotepa-fludarabine based RIC regimen, with an ex vivo and in vivo T-cell depletion performed by CD34+ cell selection and alemtuzumab infusion. Fifty-four CD8-depleted DLIs were administered to 23 pts [dose level 1 (n=4): 1×104/kg on days + 45, +75, +105; dose level 2 (n=11): 5×104/kg on days + 45, +75, +105; dose level 3 (n=8): 1×104/kg on day+ 45, 5x104/kg on days +75 and +105]. Results: At a median follow-up of 29 months, 12 pts are alive (n=3 Hodgkin Lymphoma, n=6 Non-Hodgkin Lymphoma, n=1 Multiple Myeloma, n=2 Acute Myeloid Leukemia) and 16 died from any cause [n=6 for non-relapse mortality (NRM), n=10 for disease progression] with a 2-year estimates of overall survival of 39%. The 2-year cumulative incidence of NRM and relapse were 26% and 51%, respectively. Six pts (26%) developed grade II-IV acute graft-versus-host disease (GVHD) (dose level 1: no GVHD; dose level 2: 5 cases; dose level 3: 1 case). Following CD8-depleted DLIs, the major findings were:(i) a significant expansion of memory CD4+ cells and CD19+ cells (median value 107/μL and 135/μL, respectively) at 120 days after haplo-SCT; (ii) de-novo T-cell responses to cytomegalovirus (CMV) peptides performed by activation-induced CD137 expression: the median frequency of CMV-specific CD8+/CD137+ and CD4+/CD137+ cells were 2% and 0.4% at 200 days after haplo-SCT, respectively, as evaluated in a subset of pts at risk for CMV reactivation (R CMVpos/D CMVpos/neg); this frequency was associated to a clearance of CMV antigen; (iii) recovery of thymopoiesis: 10 of 20 (50%) pts showed measurable TREC at 1 year after haplo-SCT; (iv) the increase of CD19+ cells was associated to reconstitution of B lymphocyte repertoire as analysed by Immunoglobulin heavy chain (IgH) CDR3 spectratyping: median number peaks in normal donors and pts, at day 360 after haplo-SCT, were 17 (range, 14–21) versus 12 (range, 7–14), respectively. Conclusions: Our study showed that CD8-depleted DLIs are a feasible procedure with low acute GVHD when using dose level 3. Long-term disease remissions can be observed in advanced lymphoid malignancies. Escalated doses of CD8-depleted DLIs exert complex effects on immune reconstitution: (i) provide help to expansion of CD8 T-cell clones, as suggested by the occurrence of the anti-CMV reactivity; (ii) support B-cell recovery, as demonstrated by a partial recovery of CDR3 polyclonality.

Description: Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34+ cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

Description: In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34+ selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR+ NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56bright/CD56dim NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56bright, and NKG2A and CD62L up-regulation on CD56dim, suggest sequential CD56bright-to-CD56dim NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR+ NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR+ NK cells selected from an alloreactive donor.

Description: 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 from a KIR ligand matched (n=5) or mismatched (n=6) haploidentical family donor, using high doses (median 12.5x106/kg) of purified CD34+ progenitors. Nine patients achieved long-term (〉150 days) complete remission of disease, independently from disease status at time of transplantation, and, importantly, from the presence (n=5) or absence (n=4) of donor NK alloreactivity. Within the first two months after transplantation, the vast majority (96% at 30 days, 86% at 60 days; SD 2% and 11%, respectively) of NK cells arising in the patients expressed the inhibitory receptor CD94/NKG2A, whose ligand HLA-E is ubiquitously expressed by cells positive for classical HLA class I molecules including leukemic blasts. As shown by mAb inhibition studies, lysis of patient-derived phytohemagglutinin-activated T cell blasts by these early arising NK cells was specifically inhibited by engagement of CD94/NKG2A. KIR expression was restored with variable kinetics in the later post-transplantation phase (3–9 months). Interestingly, however, during this period, NK cells devoid of CD94/NKG2A were found to express at least one KIR specific for an HLA ligand present in the patient, suggesting functional silencing of NK cells arising in the later phases after transplantation by acquisition of specific KIRs. Taken together, these data challenge current broad view on putative antileukemic effect of alloreactive NK cells reconstituting from haploidentical donor CD34+ cells and suggest that optimal exploitation of NK alloreactivity for GvL requires the presence of NK cells matured in the context of the donor’s rather than the recipient’s HLA repertoire. Ultimately, these findings provide a rationale for emerging clinical evidence in favor of efficacy of NK-based immunotherapy with mature donor NK cells.

Description: In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.