Adipose tissue-derived mesenchymal stromal cells for clinical application: An efficient isolation approach.

PURPOSE OF THE STUDY: Mesenchymal stromal cells (MSCs) are considered a promising tool for cell therapy approaches. The translation of research-based cell culture protocols into procedures that comply with Good Manufacturing Practice (GMP) is critical. The aim of this study was to design a new method for the expansion of MSCs from Adipose Tissue (AT-MSCs) in compliance with GMP, without enzymatic tissue digestion and without the use of animal proteins as source of growth factors. PATIENTS AND METHODS: MSCs were expanded from 10 periumbilical biopsies. Our new isolation approach is based on: (1) disruption of AT with an automated, closed system; (2) use of GMP-grade medium without the addition of fetal bovine serum or platelet lysate; (3) use of human recombinant Trypsin. AT-MSCs cultured in α-MEM and minced by scalpel were used as control. RESULTS: It was possible to expand MSCs from all the AT-samples for at least eight passages. MSCs displayed the typical spindle-shape morphology, a high viability, multilineage differentiation potential and high expression levels of the typical MSC-specific surface antigens and genes. Compared to standard method, MSCs obtained with the new method showed higher yield, up to passage 6, and higher purity in terms of percentage of CD34 and CD45 markers. All AT-MSCs exhibit in vitro immunosuppressive capacity and possess a normal karyotype. CONCLUSIONS: Our data clearly demonstrate that our new approach permits to generate AT-MSCs fully compliant for therapeutic use and better at least in terms of quantity and purity than those obtained with the standard method.

T-cell therapy for EBV-associated nasopharyngeal carcinoma: preparative lymphodepleting chemotherapy does not improve clinical results.

BACKGROUND: We and others have demonstrated that adoptive cell therapy with Epstein-Barr virus (EBV)-specific autologous cytotoxic T lymphocytes (CTLs) may control disease progression in patients with EBV-associated nasopharyngeal carcinoma (NPC). With the aim of favoring in vivo T-cell expansion, we optimized our cell therapy approach by administering higher doses of EBV-specific CTLs, following lymphodepleting chemotherapy. PATIENTS AND METHODS: Eleven patients with EBV-related NPC in whom conventional treatment failed have been enrolled. Patients received nonmyeloablative lymphodepleting chemotherapy consisting of cyclophosphamide and fludarabine. Two doses of autologous EBV-specific CTLs were subsequently infused, 2 weeks apart. Study end points were feasibility and clinical outcome. RESULTS: All patients enrolled completed the treatment and were assessable for analysis. The median dose of CTLs per infusion was 3.7 × 10(8). Therapy was well tolerated, with no severe adverse events ascribable to either chemotherapy or cell therapy. Disease control (defined as either tumor regression or disease stabilization lasting >4 months) was obtained in 6 of 11 patients, in keeping with previously published results. CONCLUSIONS: Our data confirm that EBV-specific CTL therapy is safe and associated with antitumor activity in patients with advanced NPC. The use of lymphodepleting chemotherapy before high-dose CTL infusion did not enhance the clinical benefit observed in our previous series.

T cell therapy for nasopharyngeal carcinoma.

Among the novel biologic therapeutics that will increase our ability to cure human cancer in the years to come, T cell therapy is one of the most promising approaches. However, with the possible exception of tumor-infiltrating lymphocytes therapy for melanoma, clinical trials of adoptive T-cell therapy for solid tumors have so far provided only clear proofs-of-principle to build on with further development. Epstein-Barr virus (EBV)-associated malignancies offer a unique model to develop T cell-based immune therapies, targeting viral antigens expressed on tumor cells. In the last two decades, EBV-specific cytotoxic T-lymphocytes (CTL) have been successfully employed for the prophylaxis and treatment of EBV-related lymphoproliferative disorders in immunocompromised hosts. More recently, this therapeutic approach has been applied to the setting of EBV-related solid tumors, such as nasopharyngeal carcinoma. The results are encouraging, although further improvements to the clinical protocols are clearly necessary to increase anti-tumor activity. Promising implementations are underway, including harnessing the therapeutic potential of CTLs specific for subdominant EBV latent cycle epitopes, and delineating strategies aimed at targeting immune evasion mechanisms exerted by tumor cells.

Co-infusion of ex vivo-expanded, parental MSCs prevents life-threatening acute GVHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation.

When compared with BMT, umbilical cord blood transplantation (UCBT) is associated with a lower rate of engraftment and delayed hematological/immunological recovery. This leads to increased risk of TRM in the early post transplantation period due to infection. Acute GVHD, although occurring less frequently in UCBT compared with BMT, is also significantly associated with increased rate of early TRM. BM MSCs are known to support normal in vivo hematopoiesis, and co-transplantation of MSCs has been shown to enhance engraftment of human cord blood hematopoietic cells in nonobese diabetic/SCID mice. In 13 children with hematological disorders (median age 2 years) undergoing UCBT, we co-transplanted paternal, HLA-disparate MSCs with the aim of improving hematological recovery and reducing rejection. We observed no differences in hematological recovery or rejection rates compared with 39 matched historical controls, most of whom received G-CSF after UCBT. However, the rate of grade III and IV acute GVHD was significantly decreased in the study cohort when compared with controls (P=0.05), thus resulting in reduced early TRM. Although these data do not support the use of MSCs in UCBT to support hematopoietic engraftment, they suggest that MSCs, possibly because of their immunosuppressive effect, may abrogate life-threatening acute GVHD and reduce early TRM.

Isolation and ex vivo expansion of bone marrow-derived porcine mesenchymal stromal cells: potential for application in an experimental model of solid organ transplantation in large animals.

Pharmacological aspecific immunosuppression, despite being widely used in solid organ transplantation recipients, is unable to completely prevent allograft rejection. It promotes the occurrence of sometimes life-threatening infections. Due to their immunosuppressive and anti- inflammatory properties, there is great interest in the therapeutic use of bone marrow (BM)-derived mesenchymal stromal cells (MSC). Large animal models play a crucial role to investigate the biological and functional properties of MSCs as novel cellular therapy. In the current study we sought to isolate expand ex vivo, and phenotypically characterize MSC derived from BM of 4 Large White 6-month-old piglets. Porcine MSC (pMSC) were characterized for their in vitro differentiation capacity. pMSC were successfully isolated from all BM samples. They showed spindle-shaped morphology and a stable doubling time on culture. They were positive for CD90, CD29, CD105, and negative for CD45 and CD11b. Furthermore, they differentiated, upon specific in vitro conditions toward adipogenic and osteogenic lineages. The optimization of methods for the isolation and characterization of pMSC may be useful to elucidate their biological and functional properties. The anatomy and physiology of the pig, which is similar to humans, make this animal model more attractive than small animals to test the safety and efficacy of MSC in the context of solid organ transplantation.

B lymphocyte subsets and their functional activity in the early months of life.

In the present study we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated natural antibody production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p less than 0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p less than 0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

Expansion of antileukaemia CTL lines and clones for adoptive cell therapy in paediatric patients given allogeneic haematopoietic stem cell transplantation.

Recurrence of the original disease remains the main cause of treatment failure in patients given allogeneic haematopoietic stem cell transplantation for either acute or chronic leukaemia. Infusion of donor lymphocytes (DLI) is useful for rescuing patients with chronic myeloid leukaemia, while this option is of limited value in patients with acute leukaemia. Moreover, DLI may cause fatal graft-versus-host disease (GvHD) or prolonged myelosuppression. A more sophisticated approach is that of generating and expanding ex vivo T-cell lines or clones able to selectively or preferentially lyse leukaemia blasts, while sparing non neoplastic targets. In this review, we will summarize the results we have obtained in vitro utilizing an approach based on the generation of leukaemia reactive cytotoxic T-lymphocytes through the use of apoptotic leukaemia cells as source of tumor antigens. Our approach proved to be feasible and effective in the experimental model for different types of leukaemia, even when the donor was HLA-disparate with the recipient. This strategy has to be tested in the clinical setting for proving its efficacy in preventing/treating leukaemia recurrence.

T cell therapy of Epstein-Barr virus and adenovirus infections after hemopoietic stem cell transplant.

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.

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia.

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.

Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation.

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.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

Successful in vitro priming of EBV-specific CD8+ T cells endowed with strong cytotoxic function from T cells of EBV-seronegative children.

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, with <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, it is feasible to activate EBV-specific CD8+ CTL with suitable characteristics for in vivo employment from EBV-seronegative children.

The immunosuppressive effect of human cytomegalovirus infection in recipients of allogeneic hematopoietic stem cell transplantation.

In immune-competent individuals, human cytomegalovirus (HCMV) infection is associated with impairment of T-cell function. Our goal was to evaluate prospectively whether clinically asymptomatic HCMV infection in allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients, treated pre emptively with ganciclovir, influences T-cell function as well. Mitogen-stimulated T-cell proliferative activity, together with cell surface markers, was tested in 49 patients on days + 30, + 45, + 60, and + 90 after alloHSCT and, additionally, in cases of positive HCMV pp65-antigenemia. HCMV infection was diagnosed in 19 patients. None of them developed HCMV disease. T-cell proliferative activity was significantly decreased on days when HCMV antigenemia was positive as compared to days without antigenemia. The number of pp65-positive cells negatively correlated with proliferative response. Comparison of patients who did experience HCMV infection with those who did not reveals significant decrease of T-cell proliferative activity observed on days + 30 and + 45, a time period when antigenemia was most frequently found to be positive, whereas no difference was detected on days + 60 and + 90. We conclude that, even clinically asymptomatic, HCMV infection has negative impact on T-cell proliferation capacity in alloHSCT recipients. However, pre emptive therapy with ganciclovir makes this immunosuppressive effect transient and restricted to the time of infection duration.

Adoptive transfer of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T cells with in vitro antitumor activity boosts LMP2-specific immune response in a patient with EBV-related nasopharyngeal carcinoma.

BACKGROUND: The outcome of patients with nasopharyngeal carcinoma (NPC) presenting as advanced-stage disease or failing conventional radio-chemotherapy is poor. Thus, additional forms of effective, low-toxicity treatment are warranted to improve NPC prognosis. Since NPC is almost universally associated with Epstein-Barr virus (EBV), cellular immunotherapy with EBV-specific cytotoxic T lymphocytes (CTLs) may prove a successful treatment strategy. Patient and methods A patient with relapsed NPC, refractory to conventional treatments, received salvage adoptive immunotherapy with EBV-specific CTLs reactivated ex vivo from a human leukocyte antigen-identical sibling. EBV-specific immunity, as well as T-cell repertoire in the tumor, before and after immunotherapy, was evaluated. RESULTS: CTL transfer was well tolerated, and a temporary stabilization of disease was obtained. Moreover, notwithstanding the short in-vivo duration of allogeneic CTLs, immunotherapy induced a marked increase of endogenous tumor-infiltrating CD8+ T lymphocytes, and a long-term increase of latent membrane protein 2-specific immunity. CONCLUSIONS: Preliminary data obtained in this patient indicate that EBV-specific CTLs are safe, may exert specific killing of NPC tumor cells in vitro, and induce antitumor effect in vivo.

Antigen-specific T cell response in infants after recombinant hepatitis B virus vaccination at birth: evaluation of T helper lymphocyte diversity.

Recombinant hepatitis B virus antigen (rHBsAg)-specific CD4+ T cell clones (TCC) were isolated and expanded from the peripheral blood of nine children vaccinated at birth against the hepatitis B (HB) virus. Four of them responded with protective antibody production (responders), three subjects were unable to produce detectable antibody levels even after revaccination (nonresponders), and two infants produced antibodies only after revaccination (slow responders). TCC were then characterized for their ability to produce cytokines known to be important for T cell expansion (interleukin-2, IL-2) and/or effector functions (IL-4, IFN-gamma, IL-10). Results demonstrated that the frequency of rHBsAg-specific TCC in the samples of nonresponders was comparable to or higher than that in the samples of responders. Nevertheless, the majority of TCC obtained from responders or from slow responders before revaccination displayed the T helper 1 (T(H1))-dominant phenotype, while the majority of TCC obtained from nonresponders were nonpolarized T lymphocytes. After revaccination, the distribution of the different T(H) subsets in slow responders was heterogeneous. Overall, our present data suggest that an absence or delay in developing an rHBsAg-specific antibody response to vaccination is not associated with the capacity to generate an Ag-specific T cell response. However, compared to responders, nonresponding infants react to the rHBsAg vaccination with a reduced capacity to expand and differentiate toward polarized T(H) cells.

Low incidence of severe acute graft-versus-host disease in children given haematopoietic stem cell transplantation from unrelated donors prospectively matched for HLA class I and II alleles with high-resolution molecular typing.

We evaluated the outcome of 63 children given haematopoietic stem cell transplantation from unrelated donors (URD-HSCT) prospectively selected using DNA high-resolution typing of both HLA class I and class II loci. Thirty patient/donor pairs (48%) were fully matched. Among the others, HSCT was performed in the presence of one (n=22), two (n=9), or three (n=2) HLA disparities. Patients had either malignant (n=46) or non-malignant (n=17) disease. In all cases, graft-versus-host disease (GVHD) prophylaxis consisted of cyclospor-in A, short-term methotrexate and pretransplant anti-thymocyte globulin. The probability of haematopoietic recovery at day 100 was 97%. Two patients experienced primary graft failure. The cumulative probability of grades III-IV acute GVHD and of extensive chronic GVHD equalled 8 and 14%, respectively. A total of 12 patients died of transplant-related complications. The probability of transplant-related mortality (TRM) at 100 and 180 days was 10 and 15%, respectively, whereas the cumulative incidence of TRM was 22%. The probability of GVHD-related mortality equalled 6% at 2.5 years. The overall and disease-free survival rates were 67 and 65%, respectively. URD-HSCT with donor selection based on high-resolution HLA typing is associated with low incidence of both severe acute GVHD and graft failure. The observed outcome is comparable to that of children transplanted from HLA-identical siblings.

Successful T-cell-depleted, related haploidentical peripheral blood stem cell transplantation in a patient with Fanconi anaemia using a fludarabine-based preparative regimen without radiation.

Haematopoietic stem cell transplantation (HSCT) represents the treatment of choice for severe bone marrow failure in patients with Fanconi anaemia (FA). When the donor is a compatible relative, the chance of being cured with an allograft is in the order of 70%. However, for FA children lacking an HLA-identical sibling, the results of HSCT from an alternative donor are less satisfactory because of a higher risk of graft rejection, graft-versus-host-disease (GVHD) and regimen-related toxicity. We report on a 12-year-old girl with FA, who was treated by T-cell-depleted (TCD) peripheral blood HSCT from her haploidentical uncle, using a novel fludarabine-based preparative regimen without radiation. She had rapid engraftment with no toxicity and no GVHD. Progressive recovery of both numbers of lymphocyte and of proliferative response to polyclonal activators occurred over time. At 18 months after transplantation, she is well with 100% donor chimerism and has recovered normal immune function.

Polyomavirus BK infection in pediatric kidney-allograft recipients: a single-center analysis of incidence, risk factors, and novel therapeutic approaches.

BACKGROUND: Although a growing body of literature regarding polyoma BK virus (BKV) infection and associated interstitial nephritis in kidney-allograft recipients is becoming available, the impact of BKV infection in the pediatric population has not been fully evaluated. METHODS: In a retrospective analysis, we performed polymerase chain reaction (PCR) assays for BKV DNA in serum and urine samples from 100 pediatric kidney-allograft recipients referred to our institution in the last 5 years. RESULTS: BKV viruria was observed in 26 of 100 patients, whereas BKV viremia was demonstrated in 5 patients. Serum creatinine was significantly higher in recipients with positive BK viremia compared with BKV DNA-negative patients (mean 2.66 vs. 1.14 mg/100 mL). Renal biopsy performed in 3 of 5 patients showed graft damage consistent with interstitial nephropathy. In the univariate analysis, negative antibody status of the recipient and the presence of mycophenolate mofetil in baseline immunosuppression were the two factors predictive of active BKV infection. CONCLUSIONS: Our study shows that BKV-associated nephropathy is a relevant complication in the pediatric kidney transplantation setting also. Identification of patients at risk of developing virus-associated nephropathy, through prospective quantification of viral load, could improve clinical outcome by allowing the use of timely preemptive therapy guided by BKV DNA levels.