HLA-haploidentical hematopoietic stem cells transplantation with regulatory and conventional T-cell adoptive immunotherapy in pediatric patients with very high-risk acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig. 6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

Improved outcome with T-cell-depleted bone marrow transplantation for acute leukemia.

PURPOSE: To eliminate the risk of rejection and lower the risk of relapse after T-cell-depleted bone marrow transplants in acute leukemia patients, we enhanced pretransplant immunosuppression and myeloablation. PATIENTS AND METHODS: Antithymocyte globulin and thiotepa were added to standard total-body irradiation/cyclophosphamide conditioning. Donor bone marrows were depleted ex vivo of T lymphocytes by soybean agglutination and E-rosetting. This approach was tested in 54 consecutive patients with acute leukemia who received transplants from HLA-identical sibling donors or, in two cases, from family donors mismatched at D-DR. No posttransplant immunosuppressive treatment was given as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Neither graft rejection nor GVHD occurred. Transplant-related deaths occurred in six (16.6%) of 36 patients in remission and in seven (38.8%) of 18 patients in relapse at the time of transplantation. The probability of relapse was .12 (95% confidence interval [CI], 0 to .19) for patients with acute myeloid leukemia and .28 (95% CI, .05 to .51) for patients with acute lymphoblastic leukemia who received transplants at the first or second remission. At a median follow-up of 6.9 years (minimum follow-up, 4.9 years), event-free survival for patients who received transplants while in remission was .74 (95% CI, .54 to .93) for acute myeloid leukemia patients and .59 (95% CI, .35 to .82) for acute lymphoblastic leukemia patients. All surviving patients have 100% performance status. CONCLUSION: Adding antithymocyte globulin and thiotepa to the conditioning regimen prevents rejection of extensively T-cell-depleted bone marrow. Even in the complete absence of GVHD, the leukemia relapse rate is not higher than in unmanipulated transplants.

Possible correlation between low antigenic drift of A(H1N1) influenza viruses and induction of HI antibodies.

This study examined whether, during a seven-year period of low A(H1N1) influenza virus antigenic drift (1988-1989 and 1994-1995, winters), humoral antibody response of elderly volunteers to influenza vaccines could suggest a lack of antibody pressure for drift. In all the years studied A/Taiwan/1/86, the A(H1N1) vaccine component, had a low ability to induce protective hemagglutination-inhibiting (HI) antibody titres (> or = 1:40). However a similar low immunogenicity was found for some of the different A(H3N2) strain variants of influenza virus, co-circulating in the same period and showing a regular extent of antigenic variations. Although our data could be at least in part explained by the type of study population (elderly and repeatedly vaccinated), postepidemic serological studies did not evidence a consistently lower ability in mounting protective immune response in elderly people as compared with younger against the influenza strains studied. Therefore, our present results did not exclude a true low immunogenicity of A/Taiwan and of some A(H3N2) influenza strains, circulating in the winters examined. This suggests that, besides the necessity to evade prior immunity, additional factors could influence the frequency of influenza viruses antigenic drifts.

Successful engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum.

Patients who undergo transplantation with haploidentical "three-loci" mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical "three-loci" incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD > or = grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.

Acute rejection of interferon-treated leukemia cells injected into lethally irradiated syngeneic mice.

Interferon (IFN) treatment of target cells can alter their susceptibility to natural resistance (NR), evidenced as in vitro 'natural killer' (NK) cell-mediated lysis or as in vivo rapid cell clearance. This paper reports the consequence of direct in vitro treatment with IFN-alpha/beta or IFN-gamma on acute rejection of leukemia cells in lethally irradiated hosts. This type of rejection has the characteristics of NR, although it is specific and genetically regulated. The data were obtained injecting intravenously FLC (FLC-745 and FLC-3C18 clones; H-2d) and EL-4 (H-2b) leukemia lines in lethally irradiated syngeneic mice and evaluating proliferation 4 days later by 125IUdR uptake. Overnight pretreatment with 100 U/ml of IFN-gamma protected tumor cells from NR-induced rejection in mice. This was evident by higher 125IUdR incorporation in spleens of mice inoculated with IFN-gamma pretreated leukemia cells, as compared to that detected in the spleens of hosts injected with untreated cells, in mice with high levels of NR, but not in hosts depressed for NR. Treatment with 1,000 U/ml of IFN-alpha/beta induced protection only of FLC-745 cells, injected in Poly I:C stimulated hosts. On the other hand, a lower 125IUdR uptake after IFN-alpha/beta incubation, as compared with control cells, was evidenced with FLC-745 and EL-4 lines inoculated in mice with normal or depressed NR. The IFN-induced alterations of leukemia cells to in vivo NR susceptibility were not associated with substantial changes of binding ability to NR effectors or of MHC-antigen expression.

Immunization of elderly volunteers with the 1988-89 inactivated whole influenza vaccine: assessment of antibody responses by haemagglutination inhibition and single radial haemolysis tests.

The immunogenicity of inactivated whole trivalent influenza vaccines (A/Taiwan/1/86 (H1N1), A/Sichuan/2/87 (H3N2), and B ijing/1/87) recommended for the 1988-89 winter season was evaluated in 236 elderly (mean age 71 years) high risk volunteers. An overall significant increase in the number of subjects with protective haemagglutination inhibiting (HI) antibodies (titer > 1:40) against vaccine components was observed after vaccination. Nevertheless, a percentage of individuals (ranging from 56% to 62%) remained without protective antibodies and the number of people showing a positive response was limited (from 32% to 41%). By the comparative analysis of the results obtained examining the presence of protective levels of antibody in the sera from 91 volunteers using HI versus the single radial haemolysis (SRH) test, we obtained evidence for a higher sensitivity of SRH technique especially against B antigen.

Immunogenicity of trivalent subunit and split influenza vaccines (1989-90 winter season) in volunteers of different groups of age.

Trivalent split or subunit influenza vaccines [A/Shangai/11/87 (H3N2), A/Singapore/6/86 (H1N1) and B/Yamagata/16/88] recommended for the 1989-90 winter season and licensed in Italy, were administered to 149 volunteers of three different age groups (elderly, middle-aged and young). Antibody production was determined in pre- and postvaccination sera by haemagglutinin inhibition test and the results were evaluated as protection and response rates. The split vaccine was more immunogenic than the subunit preparation, especially against the B virus strain. Age had no obvious impact on the degree of responsiveness to vaccination.

Effect of in vitro interferon treatment on the rapid clearance of murine leukemia cells in vivo.

Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.

[Experience with anti-hepatitis-B vaccination using a recombinant DNA vaccine, in students of dentistry at the University of Perugia]. / Esperienza di vaccinazione anti-epatite B, con vaccino a DNA ricombinante, negli studenti di odontoiatria dell'Università di Perugia.

Infection with HBV is still one of the more important professional risk for medical staff. This is particularly true for dentists the more so when they are operating in areas at medium or high prevalence of HBsAg carriers. In such situation patients often ignore their carrier state, when they know it they are not always conscious of the importance of notifying the information to their dentist. The dentists, on the other hand, don't routinely collect a detailed anamnesis of their patients. For these reasons the opportunity of being vaccinated against hepatitis B with a DNA recombinant vaccine was offered to dentistry students of the University of Perugia. All participants were to be negative for hepatitis B markers of infection including HBsAg and anti HBs. All volunteers were to have normal aminotransferase levels, be in good physical condition and give their informed written consent. 48 students turned out to be eligible for vaccination, of them 35 were males, 13 females, mean age 23 years. About two months after the third dose, all the vaccinees had seroconverted and 70.4% of them showed antibody titres between 1,000 greater than or equal to 10,000 UI/l. That indicate that very likely the antibody persistence will be long lasting (at least 5 years). Side effects, light and of short duration, were rarely seen.

Immune response to trivalent inactivated influenza vaccine in young and elderly subjects.

The antibody response (determined using the single radial haemolysis in gel technique) to inactivated whole-virion trivalent influenza vaccine [A/Leningrad/360/86(H3N2), A/Taiwan/5/87 and B/Ann Arbor/1/86], recommended for the 1987-88 winter season in Italy, in 49 elderly (age greater than or equal to 60 years) subjects was compared with the response in 23 young adult (age less than 60 years) volunteers. The subjects were prevalently healthy and a high percentage of young and old people had been repeatedly immunized against influenza in previous years. No significant differences were detected among age groups; moreover, the immune response measured by seroconversion or by a significant rise in antibody titre was constantly low.

Natural resistance in mice against Friend cells injected intravenously. III. Comparison between in vivo and in vitro passaged interferon-sensitive (745) and interferon-resistant (3Cl8) cell clones.

In vitro (FLC-Vt) or in vivo (FLC-V) passaged Friend erythroleukaemia cells of DBA/2 origin were tested for susceptibility to natural resistance (NR) in vivo or to NK cell activity in vitro. Scarcely oncogenic FLC-Vt cells were highly susceptible to in vivo NR (measured as rapid organ clearance or growth inhibition in lethally irradiated mice) or to in vitro NK attack. Conversely, highly oncogenic FLC-V cells were weakly susceptible to NR and to NK as well. These data seem to point out that natural immunity, which is up-regulated by endogenous or exogenous interferons, can play a significant role in surveillance against mouse leukaemic cells of retrovirus origin.

Natural resistance in mice against Friend leukemia cells. II. Studies with in vivo passaged interferon-sensitive and interferon-resistant cell clones.

Experiments were designed to test the presence of antitumor natural resistance (NR) in DBA/2 mice against highly oncogenic in vivo passaged histocompatible Friend leukemia cells (FLC-V). NR was measured in vivo as rapid clearance of radiolabeled cells from different organs or as growth inhibition in lethally irradiated mice. Interferon-sensitive (745) or interferon-resistant (3C18) lines were used. Organ clearance studies showed that young recipients eliminate cells more rapidly than old mice. Moreover, depressive (e.g., cyclophosphamide or carrageenen) or enhancing (e.g., poly (I:C) or Friend leukemia virus infection) agents of NR function modulate accordingly leukemia cell clearance. Similar results were obtained testing tumor growth in lethally irradiated hosts, although modulating agents were substantially less effective in this system. Both FLC-745-V and FLC-3C18-V lines were equally susceptible to NR. Therefore, these data provide further support to the hypothesis that exogenous IFN capable of suppressing the growth of both lines could act via enhancement of the NR function.