Francesco Zinno

Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia.

Fetomaternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem cell transplantation from a mismatched mother to thalassemic patient without an human leukocyte antigen-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11; 30 mg/m(2) fludarabine from day -17 to -11; 14 mg/kg busulfan starting on day -10; and 200 mg/kg cyclophosphamide, 10 mg/kg thiotepa, and 12.5 mg/kg antithymocyte globulin daily from day -5 to -2. Fourteen patients received CD34(+)-mobilized peripheral blood and bone marrow progenitor cells; 8 patients received marrow graft-selected peripheral blood stem cells CD34(+) and bone marrow CD3/CD19-depleted cells. T-cell dose was adjusted to 2 x 10(5)/kg by fresh marrow cell addback at the time of transplantation. Both groups received cyclosporine for graft-versus-host disease prophylaxis for 2 months after transplantation. Two patients died (cerebral Epstein-Barr virus lymphoma or cytomegalovirus pneumonia), 6 patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic graft-versus-host disease. These results suggest that maternal haploidentical hematopoietic stem cell transplantation is feasible in patients with thalassemia who lack a matched related donor.

Enumeration of CD34+ hematopoietic progenitor cells for clinical transplantation: comparison of three different methods.

Three different methods for determination of CD34+ cells in G-CSF-mobilized peripheral blood were compared. The methods were: the Milan/Mulhouse protocol, the ISHAGE guidelines for CD34+ cells enumeration and our own protocol. The procedure we have adopted is essentially a Milan/Mulhouse protocol-derived methodology combined with a multiparametric approach using the PAINT-A-GATE software analysis program. The samples were collected from 70 patients affected by acute leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myeloma and breast cancer who were scheduled to receive autologous PBSC transplantation. PBSC collection was performed following mobilization with subcutaneous G-CSF at 5–10 μg/kg/day. A minimum target of 2 × 106/kg CD34+ cells was considered an acceptable harvest to ensure a safe transplant. On average, three aphereses per patient were performed and a total of 204 apheresis samples were analyzed. Regression analysis of the percentage and absolute number of CD34+ cells, as calculated with each method, achieved an excellent correlation in spite of methodological differences. In fact, both CD34+dim and CD34+CD45− events were included in our gating strategy. In the setting of a triple staining associating CD34, CD38 and CD45, we identified a variable fraction of CD34+CD38+CD45− cells which would be otherwise undetected due to its CD45 negativity. To this end, we used a new technology referred to as laser-scanning cytometry (LSC) which allowed the isolation and morphological identification of CD34+CD45− cells. By comparing CD34+CD45+ and CD34+CD45− cells, we found that they share a common morphology, thus confirming the hypothesis that the latter are to be considered for CD34+ cell calculation. The median number of CD34+ cells/kg, as calculated by the three methods, was: 4.79 × 106/kg (range 1–570) for the Milan/Mulhouse protocol, 3.9 × 106/kg (range 0.8–498) for the ISHAGE one, and 5.17 × 106/kg (range 2–599) for our protocol. The median time to ANC and PLT engraftment was 11 (range 9–24) and 20 (range 10–70) days, respectively. Our protocol achieved the best correlation between CD34+ cells/kg and time to ANC/PLT recovery according to the Spearman’s rank test (r = −40 and P < 0.015 for anc, r= −46 and P = 0.005 for PLT). We conclude that (1) CD45 does not appear the ideal partner of HPCA-2 for determination of hematopoietic progenitors in mobilized peripheral blood; and (2) for clinical application, a single staining with 8G12 appears simple, reliable and feasible when rigorous procedures for sample preparation and acquisition are followed and an adequate software for multiparametric analysis is available.

T cell-depleted hla-haploidentical stem cell transplantation in thalassemia young patients

The cure for thalassemia involves correcting the genetic defect in a hematopoietic stem cell that results in reduced or absent β-globin synthesis and an excess of α-globin dimers. Intracellular precipitation and accumulation of α- dimers results in ineffective erythropoiesis and hemolytic anemia. Replacing the abnormal thalassemic marrow with allogeneic normal or heterozygous stem cells carrying the functional gene restores appropriate β-globin chain synthesis.

Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system

BACKGROUND: Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC‐A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC‐A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space.

Treg cells: Collection, processing, storage and clinical use

T regulatory cells are fundamental in the maintenance of immune homeostasis and self-tolerance. Experimental models suggest the existence of two functional types of T(reg) cells designated naturally occurring and induced. Interest in T(reg) cells increased with evidence from experimental mouse and human models demonstrating that the immunosuppressive potential of these cells can be utilized in the treatment of various pathological conditions. The existence of a subpopulation of suppressive T cells was the subject of significant controversy among immunologists for many years. T regulatory cells limit immune activation through a variety of direct and indirect interactions, many of which are yet to be determined. Fully understanding T(reg) cells biology will lead us to harnessing the capacity of these cells in order to develop strategies to prevent autoimmune disorders and tolerance to transplantation. Efficient isolation, expansion and cryopreservation strategies that comply with Good Manufacturing Practice (GMP) guidelines are prerequisites for the clinical application of human CD4+ CD25+ CD127(low) FOXP3+ regulatory T cells.

Positive immunomagnetic CD34+ cell selection in haplo-identical transplants in β-thalassemia patients: removal of platelets using an automated system

BACKGROUND AIMS Immunomagnetic CD34(+) cell selection (ICS) is utilized in autologous and allogeneic transplants. In the first case it is used to reduce the neoplastic contamination of concentrates, while in the second case it is needed to carry out a T-depletion of cell concentrates in order to reduce the incidence of graft-versus-host disease (GvHD) in patients who have undergone haplo-identical transplants. METHODS The efficacy of CliniMACS technology, after reduction of platelet contamination, incubation of monoclonal antibodies (MAb) and successive washings of concentrates, performed in 16 ICS using the standard method without reducing platelet content, was compared with the use of the automated system CytoMate, which was carried out in 46 ICS. RESULTS In the group of ICS carried out after automatic manipulation, a significant statistical difference in purity was noted (91.39% versus 83.57, P = 0.017) compared with the group of ICS carried out with the standard procedure. The same significant difference was noted in relation to the remaining percentages of CD3(+) and CD19(+) cells (2.31% versus 5.68%, P = 0.012, and 1.58% versus 2.71%, P = 0.014, respectively). Recovery of CD34+ cells overlapped in the two groups (70.49% versus 68.39%, P = 0.774). CONCLUSIONS Immunomagnetic selection carried out using the automated procedure was more efficient, producing a purer sample, more efficient T-depletion and optimal reduction of B cells, without influencing cell recovery. Furthermore, conforming to good manufacturing practice (GMP) guidelines, the entire procedure with CytoMate took place in a contamination-controlled environment.

Femoral catheters: safety and efficacy in peripheral blood stem cell collection

Central venous access is necessary in patients candidate for peripheral blood stem cell (PBSC) collection. We report our experience with a dual lumen femoral catheter (Gamcath, 11 french), initially designed for hemodialysis. We studied 147 patients and performed 488 collections after mobilization with either G-CSF alone or chemotherapy + G-CSF, when the white blood cell count exceeded 1 × 109 /L, or when a measurable population of CD34+ cells (20 / μL) was detected in peripheral blood. All patients received systemic anticoagulation with a low weight heparin and ultrasound examination was performed after the removal of the catheter. Seven patients developed thrombosis (4.7%), ten experienced hematomas at the site of catether placement (6.8%) despite prophylactic platelet transfusions, while only one patient (0.6%) had a catheter-related infection. In conclusion, the short-term use of large bore femoral catheters in setting up PBSC collection seems to be associated with minimal risk of infection and low thrombotic incidence.

Positive selection of CD34+ cells by immunoadsorption: factors affecting the final yield and hematopoietic recovery in patients with hematological malignancies and solid tumors

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 paticnts affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderlinc influence on the recovery of CD34+ cells. Forty patients received a median of 5 x 10(6) CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l in a median of 10 days whereas a PLT count above 20 x 10(9)/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide. was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content.

Immunomagnetic selection of progenitor cells from peripheral blood after thawing with an automatic system in a pediatric patient with a neuroblastoma

BACKGROUND: Immunomagnetic selection of peripheral blood progenitor cells (PBPCs) in patients with tumoral infiltration in marrow makes it possible to reduce contamination of cellular concentrates, but this procedure cannot always be used, mainly because of the low cellular count in apheresis concentrates.

Overview of T-cell depletion in haploidentical stem cell transplantation.

Graft-versus-host disease (GvHD) is a common complication of allogeneic stem cell transplantation in which functional immune cells in the transplanted graft recognise the recipient as “foreign” and mount an immunological attack. Clinically, GvHD is divided into acute and chronic forms. The acute form of the disease is normally observed within the first 100 days after transplantation1. The chronic form of GvHD normally occurs after 100 days2. However, this arbitrary distinction based on the time of onset fails to reflect the different pathophysiological mechanisms and clinical manifestations of acute and chronic GvHD. Acute GvHD can occur after day 100 in patients who received a non-myeloablative conditioning regimen or donor lymphocyte infusions. In addition, GvHD with typical clinical features of chronic GvHD can develop well before day 100 and concurrent with acute GvHD3. The National Institutes of Health consensus development project has, therefore, defined new criteria for the diagnosis, staging, and response assessment of chronic GvHD. The current consensus recommends that acute and chronic GvHD should be distinguished by clinical manifestations and not by time after transplantation. The consensus conference recognises two main categories of GvHD, each with two subcategories. The broad category of acute GvHD includes classic acute GvHD (maculopapular erythematous rash, gastrointestinal symptoms, or cholestatic hepatitis), occurring within 100 days after hematopoietic stem cell transplantation or donor leucocyte infusion. The broad category of acute GvHD also includes persistent, recurrent or late-onset acute GvHD, occurring more than 100 days after transplantation or donor leucocyte infusion; for brevity, this subcategory is henceforth designated as “late acute” GvHD. The presence of GvHD without diagnostic or distinctive chronic GvHD manifestations defines the broad category of acute GvHD. The broad category of chronic GvHD includes classic chronic GvHD, presenting with manifestations that can be ascribed only to chronic GvHD. The broad category of chronic GvHD also includes an overlap syndrome, which has diagnostic or distinctive chronic GvHD manifestations together with features typical of acute GvHD4. Donor T-cells play a fundamental role in the immunological attack on host tissues in both acute and chronic GvHD. While the cytokine production pattern of acute GvHD is mostly TH1 type, TH2 cytokines predominate in chronic GvHD5. In particular, acute GvHD is mediated by donor lymphocytes infused into the recipient, in whom they encounter tissues profoundly damaged tissues by the effects of the underlying disease, prior infections, and the transplant conditioning regimen. The allogeneic donor cells encounter a foreign environment that has been altered to promote the activation and proliferation of inflammatory cells. Thus, acute GvHD reflects an exaggerated response of the normal inflammatory mechanisms that involve donor T-cells and multiple innate and adaptive cells and mediators. Three sequential phases can be conceptualised to illustrate the complex cellular interactions and inflammatory cascades that ultimately evolve into acute GvHD: (i) activation of antigen-presenting cells; (ii) donor T-cell activation, proliferation, differentiation and migration; and (iii) target tissue destruction6. Understanding of the pathophysiology of chronic GvHD is not so advanced as that of acute GvHD. Alloreactive T-cells have been implicated in the pathogenesis; however, the precise roles of specific T-cell subsets, autoantigens, alloantigens, and B-cells, and interactions of chemokines and cytokines have not been fully elucidated. The clinical manifestations of chronic GvHD are often similar to an autoimmune process, suggesting similar pathophysiology7,8. Patients with GvHD can manifest sclerodermatous skin changes, keratoconjunctivitis, sicca syndrome, lichenoid oral mucosal lesions, oesophageal and vaginal strictures, liver disease and respiratory failure9–11. Removal of T-cells from the donor graft (T-cell depletion) offers the possibility of preventing GvHD and, thereby, reducing transplant-related morbidity and mortality. The probability of acute GvHD ≥grade 2 after HLA-identical stem cell transplantation varied from 25–60% for patients transplanted with unmanipulated grafts and from 0–35% after T-cell-depleted stem cell transplants. Approximately 30–50% of patients develop chronic GvHD after an HLA-identical sibling stem cell transplant. The incidence of chronic GvHD may be even higher after allogeneic transplantation using unmanipulated peripheral blood stem cells because of the higher number of T-cells in these grafts. Extensive chronic GvHD requires prolonged immunosuppressive treatment and is associated with a mortality of more than 50%: most of the deaths are secondary to infections resulting from severe immune dysfunction12–14. T-cell depletion reduces the risk of GvHD in patients with either HLA-matched or partially matched donors and also allows the transplantation of haploidentical stem cells without increased incidence of GvHD15. Haploidentical stem cell transplantation is a valid approach for patients at high risk of disease progression without HLA-matched donors. In a haploidentical setting, most donors will share only one HLA haplotype with the patients. These haploidentical donors are readily available within a few days, are highly motivated to donate large numbers of stem cells (parental donors) and, with respect to further adoptive cellular therapy, are available during the post-transplant course16. A number of attempts to use haploidentical T-cell-replete unmanipulated bone marrow and myeloablative conditioning were made in the past. It should be highlighted that these attempts were associated with early severe and often fatal side-effects, including multi-organ failure and pulmonary oedema, a clinical picture resembling hyperacute GvHD17. While initial attempts at haploidentical stem cell transplantation used bone marrow as the source of stem cells, the possibility of mobilising and collecting peripheral stem cells and the development of graft-engineering methods for peripheral blood stem cells has allowed the design of strategies to overcome some of the obstacles of haploidentical transplantation, such as engraftment failure and the high incidence of GvHD18. A major advantage for patients transplanted with T-cell-depleted grafts is a better quality of life because of the less morbidity caused by acute and chronic GvHD. The lower probability of transplant-related morbidity and mortality offers a larger number of patients the possibility of becoming eligible for various forms of additional immunotherapy such as donor leucocyte infusions with disease-specific cytotoxic T-lymphocytes, activated donor natural killer cells and dendritic cell vaccination strategies. An increase of the graft-versus leukemia effect without introducing significant GvHD may result in lower relapse rates and increased probabilities of leukemia-free and overall survival19–21.