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Background Donor/recipient mixed chimerism has been reported to be associated with an increased risk of graft failure in patients with β-thalassemia given a bone marrow transplant. We investigated the relationship between the degree of mixed chimerism over time and clinical outcome of children undergoing cord blood transplantation for β-thalassemia.Design and Methods Twenty-seven consecutive children given a cord blood transplant from a related donor were analyzed by short tandem repeat polymerase chain reaction and their chimerism results were compared with those of 79 consecutive patients who received a bone marrow transplant from either a relative (RD-BMT, n=42) or an unrelated donor (UD-BMT, n=37). Cord blood and bone marrow recipients received comparable preparative regimens.Results All cord blood recipients engrafted and displayed mixed chimerism early after transplantation; 13/27 converted to full donor chimerism over time, while 14 maintained stable mixed chimerism; all patients are alive and transfusion-independent. Twenty-four of the 79 bone marrow-recipients (12 UD- and 12 RD-BMT) exhibited full donor chimerism at all time points examined, 4/79 (2 UD- and 2 RD-BMT) did not engraft and 51/79 (23 UD- and 28 RD-BMT) displayed mixed chimerism at the time of hematologic reconstitution. Forty of 51 bone marrow recipients with mixed chimerism converted to full donor chimerism (17 UD- and 23 RD-BMT), 3/51 maintained stable mixed chimerism (1 UD- and 2 RD-BMT), while 8/51 (5 UD- and 3 RD-BMT) progressively lost the graft, and became transfusion-dependent again.Conclusions Mixed chimerism is a frequent event and does not predict the occurrence of graft failure in children with β-thalassemia given a cord blood transplant from a relative.

Failure to mount or maintain a protective immune response may influence the development of polyomavirus BK (BKV)-associated nephropathy (PVAN). However, limited data are so far available on BKV-specific immunity after kidney transplantation. BKV-specific cellular immune response was retrospectively analyzed in kidney recipients with or without BKV infection/reactivation by measuring the frequency of interferon (IFN)-gamma-secreting cells in peripheral blood. Patients with BKV-active infection and good renal function (n=6) had a mean BKV-specific lymphocyte frequency 2 log lower than healthy controls and in the same range as BKV-seropositive recipients without active infection (n=7). Patients with PVAN (n=5) revealed undetectable levels of BKV-specific cells. However, two patients from the latter cohort treated with immunosuppression reduction showed the emergence of specific immunity, with IFN-gamma production in the same range as healthy controls. Our preliminary data suggest that lack of protective immunity toward BKV may favor the occurrence of BKV active infection and influence the progression to PVAN.

Epstein–Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) and adenovirus (AdV)-related pathologies are life-threatening complications of immunosuppression in recipients of hematopoietic stem cell transplantation (HSCT). In certain cohorts (unrelated and haploidentical donor HSCT, T-cell-depleted allograft), the risk of developing these complications is higher. Here we describe the impact of T cell therapy, within programs of specific routine surveillance and preemptive treatment, on the course of EBV infection, and development of related disease, in pediatric recipients of T-cell-depleted, HLA-haploidentical HSCT. Future prospectives include the transfer of this technology to treat AdV-related complications following HSCT.

The treatment of Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti-CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T-cell depleted HSCT from an HLA-haploidentical relative. Twenty-seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20(-)/CD19(+) B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV-specific cytotoxic T-lymphocytes (CTLs), and persist in remission at a median 30-month follow-up. EBV-specific T-cell frequency, undetectable at time of EBV DNA positivity, was restored by T-cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo-HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV-specific CTLs. The treatment of Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti-CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T-cell depleted HSCT from an HLA-haploidentical relative. Twenty-seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20-/ CD19+ B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV-specific cytotoxic T-lymphocytes (CTLs), and persist in remission at a median 30-month follow-up. EBV-specific T-cell frequency, undetectable at time of EBV DNA positivity, was restored by T-cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo-HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV-specific CTLs. © 2007 The Authors.

Epstein-Barr virus (EBV)-seronegative transplant recipients are at high risk of developing EBV-associated post-transplant lymphoproliferative disorder (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. So far, efforts to activate CD8+ EBV-specific cytotoxic T lymphocytes (CTL) endowed with high specific cytotoxicity from EBV-seronegative children have failed. We compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on lymphoblastoid cell lines (LCL) stimulation potentially enhanced by either LCL presentation through dendritic cells, or selection of IFN-gamma+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, according to the standard protocol for reactivation of EBV-specific CTL. We found that only specific LCL stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children. The lines thus activated, which included specificities toward EBV latent and lytic proteins, showed high percentage CD8+ T cells, with10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7 T-cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, it is feasible to activate EBV-specific CD8+ CTL with suitable characteristics for in vivo employment from EBV-seronegative children.

Purpose Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) –related malignancy expressing EBV antigens that are possible targets of cell therapy, including latent membrane protein 2 (LMP2). We conducted a clinical trial of EBV-targeted cell therapy with autologous virus-specific cytotoxic T lymphocytes (CTLs) for NPC refractory to conventional treatments. Patients and Methods Ten patients with EBV-related stage IV NPC in progression after conventional radiotherapy and chemotherapy received intravenously autologous EBV-specific CTLs reactivated and expanded ex vivo from peripheral blood lymphocytes through stimulation with EBV-transformed autologous B-lymphoblastoid cell lines (LCL). Toxicity, specific cellular immune responses, and clinical tumor responses were evaluated. Results EBV-specific CTLs could be generated in all patients and were predominantly CD3+/CD8+ T lymphocytes displaying specific killing of autologous EBV-LCL, autologous NPC cells as well as autologous targets bearing the EBV antigen LMP2. Patients received two to 23 infusions of EBV-specific CTLs that were well tolerated with the exception of grade 1 to 2 inflammatory reactions at the tumor site in two cases. Control of disease progression was obtained in six of 10 patients (two with partial response and four with stable disease). Analysis of interferon-γ–producing cells demonstrated an increased frequency of EBV-specific immunity, with appearance of LMP2-specific responses in four patients, of whom three had clinical benefit. Conclusion Cell therapy with EBV-targeted autologous CTLs is safe, induces LMP-2-specific immunologic responses, and is associated with objective responses and control of disease progression in patients with stage IV NPC resistant to conventional treatments.

Epstein-Barr virus (EBV)–associated posttransplantation lymphoproliferative disorders (PTLDs) are a well-recognized complication of immunosuppression in solid organ transplant recipients. The reported therapeutic approaches are frequently complicated by rejection, toxicity, and other infectious pathologies, and overall mortality in patients with unresponsive PTLD remains high. Thus, low-toxicity treatment options or, preferably, some form of prophylactic/preemptive intervention are warranted to improve PTLD outcome in this setting. We assessed whether transfer of EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from the peripheral blood of allograft recipients receiving immunosuppression could increase EBV-specific killing in vivo without augmenting the probability of graft rejection. Autologous EBV-specific CTLs were generated for 23 patients who were identified as being at risk of developing PTLD through the finding of elevated EBV DNA load. Of the 23 patients, 7 received 1 to 5 infusions of EBV-specific CTLs. CTL transfer was well tolerated, and none of the patients showed any evidence of rejection. An increase of the EBV-specific cytotoxicity was observed after infusion, notwithstanding continuation of immunosuppressive therapy. EBV DNA levels had a 1.5- to 3-log decrease in 5 patients, whereas in the other 2 graft recipients CTL transfer had no apparent stable effect on EBV load. Our data suggest that the infusion of autologous EBV-specific CTLs obtained from peripheral blood mononuclear cells recovered at the time of viral reactivation is able to augment virus-specific immune response and to reduce viral load in organ transplant recipients. This approach may, therefore, be safely used as prophylaxis of EBV-related lymphoproliferative disorders in these patients, following a strategy of preemptive therapy guided by EBV DNA levels.

Allogeneic cord blood transplantation (CBT), especially from unrelated donors, is being increasingly used for treating paediatric patients with both malignant and non-malignant disorders. Recent clinical and experimental evidence suggests that human cord blood mononuclear cells (CBMC) may acquire in utero a state of tolerance towards non-inherited maternal antigens (NIMA). In order to better define this phenomenon, we measured, by means of a limiting dilution assay (LDA), the frequency of NIMA-specific CTL precursors (CTLp) in cord blood samples obtained from 13 healthy neonates. The immunophenotype of the effector cells recovered from LDA was also analysed. Data concerning both CTLp frequency and phenotype of effector cells were compared with those obtained stimulating CBMC with cells of paternal origin (NIPA) and adult PBMC with allogeneic targets. Results showed that cytotoxic cells directed towards cells of maternal origin could be detected in all cord blood samples tested. Phenotype analysis demonstrated that NIPA elicit the expansion of CD3+/CD8bright T cells, a phenotype associated with alloreactive CTL. By contrast, NIMA preferentially stimulated the expansion of CD3-/CD8dim+ cells, a phenotype associated with NK cells, which are known to be able, in certain clinical conditions, to kill allogeneic haematopoietic cells without causing GVHD. Thus, our results indicate that, when evaluated in a limiting dilution condition, NIMA-reactive cord blood cells are detectable and a preferential expansion of NK cells is observed.

Allogeneic cord blood transplantation (CBT) is increasingly used in children with beta-thalassemia major. Donor/recipient mixed chimerism (MC) is frequently observed in patients with beta-thalassemia major given bone marrow transplantation (BMT) in the early post-transplant period, due to the persistence of haematopoietic and immune cells of host origin. Previous clinical data reported on these patients demonstrated that the majority of them exhibiting MC convert to full donor chimerism (FDC), some maintain MC stable over time, while a non negligible number of patients develop graft failure, in particular when increasing proportions of cells of host origin are observed in serial monitoring. Aims of the present study were to retrospectively investigate the proportion of children with beta-thalassemia major experiencing either transient, or long-lasting persistence of hematopoietic cells of host origin after CBT, as compared to those given BMT, and to analyze the relationship existing between the evolution of MC over time and clinical outcome. One hundred and six consecutive patients, 27 given CBT from an HLA-identical related donor (RD) and 79 given BMT (42 from a RD and 37 from an HLA-compatible unrelated volunteer, UD), were enrolled in the study. Donor/recipient chimerism status was evaluated by PCR-based assay, analyzing selected polymorphic short tandem repeat (STR) loci. CBT and BMT recipients received similar preparative regimens. Anti-thymocyte globulin (ATG 10 mg/Kg on days −4, −3 and −2; Fresenius, Graefelfing, Germany) was used in all patients given an UD-BMT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (Cs-A) alone in CBT recipients; either CsA alone (12 RD-BMT recipients) or CsA in combination with methotrexate (MTX) was given to BMT patients. All patients given CBT engrafted and displayed MC early after transplantation, 13/27 converted to FDC (6 within 1 month and 7 within 1 year after CBT), 14/27 maintained stable MC, and all of them are alive, transfusionin-dependent and free of any immune-suppressive drug, the follow-up ranging between 15 and 89 months (median value 42 months). In the group of children given BMT, 24/79 (12 transplanted from UD and 12 from RD) exhibited FDC at all time points examined, 4/79 (1 transplanted from UD and 3 from RD) did not engraft and 51/79 (23 from UD and 28 from RD) displayed MC at time of haematological reconstitution. Forty out of these 51 BMT recipients with MC status converted to FDC (17 from UD and 23 from RD; 29 within 1 month and 11 within 1 year after BMT), 3/51 maintained stable MC (1 undergoing UD- and 2 RD-BMT), while 8/51 (5 from UD and 3 from RD) progressively lost the graft, returning to be transfusion-dependent. The median follow-up time was 51 months (range: 15–110 months) and 48 months (range: 16–81 months) in the RD-BMT and UD-BMT group, respectively. None of the patients given CBT experienced either grade II-IV acute GVHD, which was, by contrast, diagnosed in 3 and 7 patients given RD- and UD-BMT, respectively. Chronic GVHD was diagnosed in 8 children (2 from RD- and 6 from UDBMT). Five out of 106 pediatric patients enrolled in the study, 2 included in the RD-BMT and 3 in the UD-BMT group, died of transplantation-related complications. These results indicate that MC is frequently observed in children with beta-thalassemia after RD-CBT, without, however, predicting an increased risk of treatment failure. CBT from an HLA-identical related donor is a successful option in beta-thalassemia major, possibly also due to the establishment of an effective state of immune tolerance between host and donor that decreases the risk of both graft rejection and GVHD.

EBV-seronegative solid organ transplant recipients, or recipients of T-cell depleted hematopoietic stem cell from a EBV-seronegative donor, are cohorts at high risk for the development of EBV-associated posttransplant lymphoproliferative disease (PTLD), and would maximally benefit from an EBV-directed T-cell therapy for prevention or treatment of PTLD. Reported experiences have shown that CTL obtained from EBV-seronegative individuals are almost exclusively HLA-class II-restricted CD4+ cytotoxic T cells, and efficiency of this CTL subset in the control of PTLD has not yet been demonstrated in vivo. Conversely, the central role of CD8+ CTL in the cell-mediated containment of EBV infection is well established. Therefore, we endeavoured to develop a protocol of EBV-CTL priming that would allow optimal expansion and high specific cytotoxic activity of CD8+ CTL. We employed naïve lymphocytes obtained from 5 EBV seronegative patients with end-stage renal failure receiving dialysis while listed for a kidney transplantation (4 children, 1 adult). In particular, we compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on LCL stimulation potentially enhanced by either LCL presentation through DC, or selection of IFNg+ cultured cells, or culture in the presence of rhIL-12 and rhIL-7, to the standard protocol for reactivation of EBV-specific CTL from EBV-seropositive individuals. We found that while all protocols employed, with the exception of IFN-g+ T cell selection, allowed for the amplification of CD8+ and CD4+ mediated EBV-specific cytotoxic T cell responses in the EBV-seronegative adult, only specific EBV-transformed lymphoblastoid cell lines (LCL) stimulation in the presence of rhIL-12 and rhIL-7 was able to reproducibly generate and expand EBV-specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV-seronegative children (mean lysis at a E:T ratio of 10:1: 14±10% with the standard protocol vs. 66±30% with the addition of rhIL-7 and rhIL-12). The lines thus activated showed a higher percentage CD8+ T cells, with less than 10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7- T cell effectors was comparable to that observed in healthy EBV-seropositive controls. In conclusion, stimulation with LCL in the presence of IL-12 and IL-7 activates EBV-specific, highly cytotoxic CD8+ CTL from EBV-seronegative subjects, this opening new perspectives for prevention/treatment of PTLD.

SUMMARYExpression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8·04 ± 4·87%; p75+ 28·27 ± 18·85%) compared with healthy controls (p55+ 7·26 ± 6·17%; p75+19·42 ± 10·49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76–90%) of p75+ PBL, which were mostly CD3− and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. rL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.