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.

CD26 surface molecule involvement in T cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis.

T cell surface expression and the functional role of CD26 antigen (Ag), a surface ectoenzyme involved in T cell activation and migration across the extracellular matrix, were analyzed in the peripheral blood (PB) and synovial fluid (SF) from patients with inflammatory arthritides. CD26 membrane expression on T cells was detected by cytofluorometry using two different monoclonal antibodies, anti-Ta1 and anti-1F7, while cell proliferation and both IL-2 and IFN-gamma production were evaluated in anti-CD3- or anti-CD2-stimulated cell cultures after Ag surface modulation with anti-1F7. The results showed that Ta1 and 1F7 Ag expression were increased on T cells from PB of patients with active, but not inactive, rheumatoid arthritis (RA). Most SF T cells from RA or other inflammatory arthritides displayed the memory marker CD45R0 and the Ta1 Ag, but lacked the 1F7 molecule. In addition, in vitro 1F7 modulation, which enhanced RA PB T cell proliferation and both IL-2 and IFN-gamma synthesis, did not synergize with anti-CD3 or anti-CD2 in inducing IL-2-dependent activation of SF T cells, but reduced IFN-gamma production. A spontaneous reappearance of 1F7 Ag on the SF T cell surface was seen after 2-5 days in culture. Phorbol myristate acetate, able to accelerate its reexpression, also restored a normal response of SF T cells to anti-1F7 comitogenic effects. These data confirm a role of the CD26 surface molecule in regulating T cell activation and lymphokine synthesis. This observation may have important implications in the regulation of T cell activity at the joint level during chronic inflammatory processes.

Natural killer (NK)-cell function and antileukemic activity of a large population of CD3+/CD8+ T cells expressing NK receptors for major histocompatibility complex class I after "three-loci" HLA-incompatible bone marrow transplantation.

We have shown that addition of granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells to the marrow inoculum allows engraftment of T-cell depleted, "three loci" HLA-incompatible marrow transplants for acute leukemia. The event-free survival of patients at high risk for potential of this transplant. Tumor-cell lysis by natural killer (NK) cells is regulated by inhibitory receptors for specific HLA class I alleles. Here, we report the postgrafting emergence of a large, donor-type CD3+/CD8+ T-cell receptor (TcR)-alpha beta+ cell population, barely detectable in normal subjects, that expresses 58 kD, "p58," NK receptors for HLA-C locus alleles. Analysis of > 900 clones revealed that 40% to 80% of these T cells exhibit NK-like function, i.e., they lysed class I- targets and were functionally blocked by class I alleles on target cells. Monoclonal antibody-mediated blocking of class I recognition by these cells induced lysis of HLA-protected, autologous targets. The class I-mediated inhibitory signaling through the NK receptors also blocked TcR/CD3-triggered cytotoxicity of these cells, indicating that their antigen-specific responses may be impaired. However, the NK-like function of these cells allows them to discriminate normal cells, protected from lysis, from leukemic cells that were lysed and may be targets for a graft-versus-leukemia effect.

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.

TGF-beta is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans.

Resistance and susceptibility of mice to systemic infection with the fungus Candida albicans are associated with the preferential expansion of Th1 and Th2 cells, respectively. In this study, endogenous production of TGF-beta was found to be increased soon after infection of healer mice with a live vaccine strain of the fungus, but down-regulated in nonhealer mice with virulent yeast challenge. Although not affecting the outcome of primary challenge, serologic ablation of TGF-beta in the former animals abrogated development of acquired resistance and resulted in impaired production of IL-12/IFN-gamma and higher expression of IL-4/IL-10 at the time of reinfection with virulent yeast. A CD4+ population expressing the memory phenotype, CD44highMEL-14low, which appeared to be expanded by yeast infection of nonhealer mice, was similarly increased in the healer mice by anti-TGF-beta treatment. In vitro rTGF-beta impaired the candidacidal function of IFN-gamma-activated macrophages. Yet in nonhealer mice infected with virulent C. albicans, administration of rTGF-beta delayed progression of the disease, which was concomitant with the detection of lower levels of IL-4. In addition to previous evidence for an obligatory role of IFN-gamma and IL-12 in Candida-driven Th1 cell differentiation in vivo, the present data establish TGF-beta as a third cytokine, the presence of which may be required for optimal Th1 development leading to long-lived anticandidal resistance.

Expression and functional role of 1F7 (CD26) antigen on peripheral blood and synovial fluid T cells in rheumatoid arthritis patients.

The expression and the functional role of the CD26 (1F7) T cell surface molecule, an ectoenzyme which seems to represent a functional collagen receptor of T lymphocytes and to have a role in T cell activation, were analysed in both peripheral blood (PB) and synovial fluid (SF) T cell samples from patients with active and inactive rheumatoid arthritis (RA). Although patients with active disease displayed higher percentages of PB CD26+ CD4+ T cells than inactive RA and control subjects, CD26 antigen expression on RA SF T lymphocytes was low. The anti-1F7 binding to the T cell surface, that led to CD26 antigen modulation and enhancement of both IL-2 synthesis by, and 3H-TdR incorporation of, anti-CD3- or anti-CD2-triggered PB T cells in RA and control subjects, was unable to affect significantly both expression and functional activity of RA SF T lymphocytes. Since the 1F7 antigen spontaneously reappeared on the surface of unstimulated SF T cells after 2-5 days of culturing, the low 1F7 antigen expression of anti-1F7 in the SF T cell compartment may be the result of in vivo molecule modulation exerted by the natural ligand in the joint, with important implications for T cell activation and lymphokine synthesis.

Activation of cord T lymphocytes. IV. Analysis of surface expression and functional role of 1F7 (CD26) molecule.

A role for CD26 surface antigen (Ag) in both CD3- and CD2-mediated T cell activation has been previously demonstrated. To analyze the functional role of CD26 in the CD3- and CD2-induced activation pathways of cord T cells, which represent the most reliable source of Ag-unprimed T cells, we employed a newly developed anti-CD26 monoclonal antibody, termed anti-1F7, anti-CD3 and anti-CD2 in activating T lymphocytes. The results showed that CD26 Ag is expressed on the surface of almost all resting cord T cells and that its fluorescence intensity is enhanced by activation. The binding of anti-1F7 induced a decrease in CD26 membrane expression, with no detectable effect on the surface expression of other cord T cell-related molecules. Moreover, the modulation of CD26 resulted in an increase in anti-CD3-mediated cord T cell activation through an enhancement in intracellular calcium levels, IL-2 receptor expression, and IL-2 synthesis, whereas it had no effect on cord T cell activation induced by anti-CD2 or anti-CD2 plus exogenous IL-2. The fact that the selective involvement of CD26 in the activation pathway triggered by anti-CD3, but not anti-CD2, could be reversed by prior stimulation of cord T cells with anti-CD3 suggests that this functional feature, which resembles that of mature thymocytes, may be linked to the Ag-unprimed cell phenotype of cord T lymphocytes.

Activation of cord T lymphocytes. III. Role of LFA-1/ICAM-1 and CD2/LFA-3 adhesion molecules in CD3-induced proliferative response.

As cord T cells, a model of antigen (Ag)-unprimed cell, display a functional defect when stimulated through the CD3 molecule, the role of lymphocyte function-associated antigen 1(LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and CD2/lymphocyte function-associated antigen 3 (LFA-3) receptor-ligand pairs in cord CD3-triggered T-cell activation was analyzed using specific monoclonal antibodies (mAb) against each adhesion molecule. The addition of anti-CD11a, anti-CD18, or anti-CD2 to both adult and cord peripheral blood mononuclear cells (PBMC) cultures led to a decrease in CD3-induced proliferation. In contrast, CD3-stimulated cord, but not adult, PBMC proliferation was markedly enhanced when anti-CD54 or anti-CD58 were added. Despite the fact that ICAM-1 and LFA-3 molecules were virtually absent on cord resting T cells, mAb against these two molecules boosted both mitogenesis of and interleukin (IL)-2 production by purified cord T cells stimulated with plastic immobilized anti-CD3. Cord T-cell supernatant levels of interferon-gamma (IFN-gamma) were undetectable with CD3 stimulation, slightly raised with CD58/CD3 costimulation, but normal when T cells were preincubated with IL-2 for 24 hr before being costimulated with anti-CD3/CD58. Evidence that IL-2 and IFN-gamma play a pivotal role in fully activating cord T cells came from the demonstration that IL-2 and IFN-gamma are able to bypass the CD3-proliferative defect through differential up-regulation of the adhesion molecules. It would, therefore, seem that ICAM-1 and LFA-3 molecules are crucially implicated in the CD3-activation pathway of Ag-unprimed T cells.

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells.

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR alpha/beta+ clones, and V delta 1 or V delta 2 TCR Y/delta+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells.

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR α/ß+ clones, and Vδ1 or Vδ2 TCR Y/δ+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.

Inhibition of human breast cancer cell (MCF-7) growth in vitro by the somatostatin analog SMS 201-995: effects on cell cycle parameters and apoptotic cell death.

Somatostatin (SS) and SS analogs have been shown to exert an antiproliferative effect on several transplantable tumors in animals and to reduce the growth of pancreatic, pituitary, and mammary tumor cells in vitro. We evaluated the effects that the SS analog SMS 201-995 exerts on growth, cell-cycle parameters, and suicidal cell death (apoptosis) of human breast cancer cells (MCF-7) in vitro. SMS 201-995 significantly reduced the MCF-7 cell growth induced by serum, estradiol, insulin, and insulin-like growth Factor-I in both short term and long term experiments. The effect was maximal when 10 nM estradiol was used as mitogen in long term cultures. SMS 201-995 treatment produced a slight but transient accumulation of cells in the G2/M phase but did not cause any noteworthy reduction in the percentage of proliferating cells. There was, instead, a time-related increase in the number of cells with the flow-cytometric characteristics of apoptosis in the cultures treated with the SS analog, which correlated well with its growth-inhibiting activity. It would, therefore, seem that SMS 201-995 exerts its inhibitory effect on MCF-7 cell growth in vitro mainly by enhancing the rate of programmed (or suicidal) cell death in the culture.

Analysis of T cells bearing different isotypic forms of the gamma/delta T cell receptor in patients with systemic autoimmune diseases.

The expression of gamma/delta T cell receptor (TCR) on peripheral blood CD3+ cells circulating in 74 patients with different systemic autoimmune diseases was evaluated. There was a significant increase in the gamma/delta T cell number only in patients with primary Sjögren's syndrome (SS) and in untreated patients with systemic lupus erythematosus (SLE). Unlike healthy subjects, a subgroup of patients with SLE and SS displayed a marked increase in gamma/delta T cells. Immunosuppressive treatment of patients with active SLE led to a normalization of the gamma/delta T cell number. Analysis of surface phenotype showed that when patient gamma/delta T cells were expanded in the peripheral blood, they were not activated but bore "memory" markers. In addition, they preferentially expressed the disulfide linked form of the TCR, except in progressive systemic sclerosis where the nondisulfide form was displayed. Serial determinations in single patients demonstrated that the gamma/delta T cell increase is a persistent immunological feature in these patient subgroups.

[Pathogenetic and clinical significance of the adhesion molecule expression on T cells of the lung in sarcoid alveolitis]. / Significato patogenetico e clinico dell'espressione di molecole di adesione su cellule T del polmone durante l'alveolite sarcoidea.

A double immunofluorescence analysis of CD4+ cell population from bronchoalveolar lavage (BAL) fluid samples of patients with active pulmonary sarcoidosis was carried out. The results showed that, unlike BAL and peripheral blood CD4+ cells of healthy subjects, almost all BAL CD4+ cells of the patients highly express, besides CDw29 antigen, LFA-1 and ICAM-1 adhesion molecules. The co-expression of these molecules on BAL CD4+ cells during high intensity sarcoid alveolitis could represent a marker of immunological memory. The relevant pathogenetic and clinical implications of this observation are discussed.