T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Identification of a subpopulation of lymphocytes in human peripheral blood cytotoxic to autologous fibroblasts.

A naturally occurring subpopulation of human peripheral blood lymphocytes is cytotoxic to autologous and/or allogeneic fibroblasts. The autocytotoxic lymphocytes have a receptor for the third component of complement and for aggregated gamma globulin, do not form rosettes with sheep red blood cells, and are not removed by passage through nylon. The autocytotoxic subpopulation is not present in the thymus and tonsils of normal children or in the peripheral blood of individuals with X-linked agammaglobulinemia. Fibroblast absorption experiments demonstrate that the autocytotoxic cells are "sensitized" to antigens expressed on allogeneic fibroblasts in addition to the antigens expressed on autologous cells. Some normal individuals have a second subpopulation of lymphocytes that may "regulate" the autocytotoxic cells. The relevance of these observations to the murine autocytotoxic cells is discussed.

Altered O-glycan synthesis in lymphocytes from patients with Wiskott-Aldrich syndrome.

The only molecular defect reported for the X-linked immunodeficiency Wiskott-Aldrich syndrome (WAS) is the abnormal electrophoretic behavior of the major T lymphocyte sialoglycoprotein CD43. Since the 70 to 80 O-linked carbohydrate chains of CD43 are known to influence markedly its electrophoretic mobility, we analyzed the structure and the biosynthesis of O-glycans of CD43 in lymphocytes from patients with WAS. Immunofluorescence analysis with the carbohydrate dependent anti-CD43 antibody T305 revealed that in 10 out of the 12 WAS patients tested increased numbers of T lymphocytes carry on CD43 an epitope which on normal lymphocytes is expressed only after activation. Other activation antigens were absent from WAS lymphocytes. Western blots of WAS cell lysates displayed a high molecular mass form of CD43 which reacted with the T305 antibody and which could be found on in vivo activated lymphocytes but was absent from normal unstimulated lymphocytes. To examine the O-glycan structures, carbohydrate labeled CD43 was immunoprecipitated and the released oligosaccharides identified. WAS lymphocyte CD43 was found to carry predominantly the branched structure NeuNAc alpha 2----3Gal beta 1----3 (NeuNAc alpha 2----3Gal beta 1----4G1cNAc beta 1----6) GalNAcOH whereas normal lymphocytes carry the structure NeuNAc alpha 2----3Gal beta 1----3 (NeuNAc alpha 2----6) GalNAcOH. Only after activation NeuNAc alpha 2----3Gal beta 1----3 (NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6) GalNAcOH becomes the principal oligosaccharide on CD43 from normal lymphocytes. Analyzing the six glycosyltransferases involved in the biosynthesis of these O-glycan structures it was found that in WAS lymphocytes high levels of beta 1----6 N-acetyl-glucosaminyl transferase are responsible for the expression of NeuNAc alpha 2----3Gal beta 1----3 (NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6) GalNAcOH on CD43. The gene responsible for WAS has not yet been identified but the results presented in this study suggest that the primary defect in WAS may affect a gene which is involved in the regulation of O-glycosylation.

Characterization of a human lymphocyte surface sialoglycoprotein that is defective in Wiskott-Aldrich syndrome.

gpL115 is a lymphocyte surface component that is deficient in patients with the X-chromosome-linked immune deficiency Wiskott-Aldrich syndrome (6). The glycoprotein nature of gpL115 is demonstrated through labeling in carbohydrate moieties by [3H]NaBH4 and its synthesis by lymphocytes through labeling with [35S]methionine. Native gpL115 adheres to wheat germ lectin-Sepharose and sialidase-treated gpL115 does not adhere, indicating that native gpL115 adheres via clusters of sialic acid residues. When tested on peanut lectin, which shows specificity for the disaccharide Gal beta 1-3GalNAc, gpL115 is nonadherent and sialidase-treated gpL115 is adherent, indicating the presence of the sequence sialic acid-Gal beta 1-3GalNAc, which is characteristic for O-linked (mucin-type, acidic-type) carbohydrates. A surface glycoprotein with all the above characteristics was found on the lymphoblastoid cell line CEM. CEM cells were used as immunogen to generate the monoclonal antibody L10, an IgG1, which binds native and sialidase-treated gpL115 . Sialidase-treatment of gpL115 significantly alters its physical properties, reducing its electrophoretic mobility and changing its behavior on isoelectrofocusing. Cumulatively, these findings indicate that gpL115 , like glycophorin of erythrocytes and GPIb of platelets, is a sialoglyco protein with significant quantities of O-linked carbohydrate. On treatment with limiting sialidase concentrations, gpL115 of normal lymphocytes is transformed into a series of partially desialylated species of decreasing electrophoretic mobility. This finding resembles the situation with lymphocytes of some Wiskott-Aldrich syndrome patients. Lymphocytes of eight Wiskott-Aldrich syndrome patients were found to be deficient in 125I-labeled gpL115 . Lymphocytes from three of these patients displayed an abnormal 125I-component of apparent mol wt 135,000.

High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.

Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.

Study of thymic size and function in children and adolescents with treatment refractory systemic sclerosis eligible for immunoablative therapy.

Following hematopoietic stem cell transplantation (HSCT), thymic reconstitution of peripheral T lymphocytes is essential to avoid a chronically immunodeficient state and disease recurrence. The purpose of this study was to determine if children and adolescents with treatment refractory SSc, awaiting HSCT, have sufficient thymic function to reconstitute T lymphocyte function after transplantation. Thirteen children with systemic scleroderma were enrolled and assessed by physical exam, chest MRI, measurement of autoantibodies, B and T cell immuno-phenotyping, and quantization of T cell receptor rearrangement excision circles (TREC) as a marker of thymopoiesis. MRI detected thymic tissue in 9/13 children. TREC levels were detectable in all but one child but were significantly reduced (p<0.001) when compared to a control population. SSc patients also had a reduced percentage of naïve (CD45RA+CD31+) CD4+ T lymphocytes, further indicating diminished thymopoiesis. Our data suggest that thymic function in children with SSc might be insufficient for an adequate immunoreconstitution following transplantation in some patients. A thorough evaluation of immune and thymic functions to identify those patients prior to HSCT is recommended.

The application of bone marrow transplantation to the treatment of genetic diseases.

Genetic diseases can be treated by transplantation of either normal allogeneic bone marrow or, potentially, autologous bone marrow into which the normal gene has been inserted in vitro (gene therapy). Histocompatible allogeneic bone marrow transplantation is used for the treatment of genetic diseases whose clinical expression is restricted to lymphoid or hematopoietic cells. The therapeutic role of bone marrow transplantation in the treatment of generalized genetic diseases, especially those affecting the central nervous system, is under investigation. The response of a generalized genetic disease to allogeneic bone marrow transplantation may be predicted by experiments in vitro. Gene therapy can be used only when the gene responsible for the disease has been characterized. Success of gene therapy for a specific genetic disease may be predicted by its clinical response to allogeneic bone marrow transplantation.

Biosynthesis of C4 (fourth component of complement) by hybrids of C4-deficient guinea pig cells and HeLa cells.

Peritoneal exudate cells from guinea pigs homozygous for a genetic deficiency of the fourth component of complement (C4) were fused in vitro with a cell line of human origin (HeLa). The resulting hybrid cells, derived from cell lines each incapable of C4 synthesis by themselves, synthesized functionally active human C4.

Murine sarcoma virus: the question of defectiveness.

Infection of mouse and rat cells by the murine sarcoma virus (Moloney isolate) showed two-hit kinetics for focus production in mouse cells but one-hit kinetics in rat cells. Antiserum added to cultures after infection suppressed focus formation in mouse cells but not in rat cells. These studies suggest that, in rat cells infected with murine sarcoma virus, leukemina virus is not needed for focus formation and that these foci result from proliferation of the transformed rat cell; in mouse cells, on the other hand, leukemia virus is needed as "helper," and focus formation requires spread of virus. The term "defectiveness" then, if used, should not be applied to RNA tumor viruses without qualification for the viral function studied and the cell system employed.

Molecular heterogeneity of a lymphocyte glycoprotein in immunodeficient patients.

Previous evaluation of lymphocytes taken from patients with Wiskott-Aldrich syndrome (WAS) and other X-linked immunodeficiencies has revealed deficiencies of a lymphocyte sialoglycoprotein with a relative molecular mass of 115 kD (designated gpL-115) found in normal lymphocytes. The development of monoclonal antibodies to gpL-115 has permitted the detection of molecular heterogeneity in gpL-115 from the lymphocytes of immunodeficient patients. When lymphocytes from normal individuals were analyzed by immunoblotting, gpL-115 with only a single molecular species (115 kD) was detected. Lymphocytes from 17 immunodeficient patients were analyzed after overnight incubation. Two patients had no gpL-115 with an Mr of 115 kD, but gpL-115 with an Mr of either 95 or 135 kD was detected. Nine patients had gpL-115 with Mr equally of 95 and 115 Kd. Other patients exhibited gpL-115 with combinations of 95, 115, and 135 kD. The heterogeneity of the degraded gpL-115 suggests that WAS and other X-linked immunodeficiencies are due to a series of abnormalities, all of which involve gpL-115, and may explain the clinical heterogeneity of the diseases.

Regulation of human immunoglobulin E synthesis in acute graft versus host disease.

Immunoglobulin (Ig) E synthesis was studied in vitro in eight patients who had received transplants of allogeneic bone marrow. Seven of these patients developed acute graft vs. host disease (GVHD) and elevated serum IgE levels, whereas the eighth did not. In vitro synthesis of IgE, but not of IgG, was elevated in cultures of lymphocytes obtained during acute GVHD (17,923 +/- 14,607 pg/10(6) cells) but not in cultures of lymphocytes obtained after resolution of the acute GVHD when the serum IgE had returned to normal (106 +/- 31 pg/10(6) cells). In contrast, lymphocytes from the patient with no acute GVHD, like normal lymphocytes, failed to synthesize IgE in vitro. The increased in vitro IgE synthesis in acute GVHD was suppressed by normal allogeneic lymphocytes and by autologous lymphocytes obtained after the resolution of the acute GVHD, but not by allogeneic lymphocytes obtained from patients undergoing acute GVHD. The deficiency in functional IgE-specific suppressor cells in acute GVHD occurred in the face of normal or increased percentages of circulating T8+ cells, which in normal subjects contain the IgE-specific suppressor cells. In two patients studied, there was evidence of activated IgE-specific, circulating helper T cells. T cells from these two patients, but not normal T cells, secreted spontaneously upon culture in vitro a factor that induced IgE, but not IgG, synthesis by normal B cells. Finally, a survey of 21 bone marrow transplant recipients revealed that acute GVHD was a necessary requirement for the development of elevated serum IgE levels in recipients of bone marrow transplants. These results suggest that acute GVHD is accompanied by an imbalance in IgE-specific immunoregulatory T cells consisting of activated helper T cells and deficient suppressor cells.

T lymphocyte ontogeny in adenosine deaminase-deficient severe combined immune deficiency after treatment with polyethylene glycol-modified adenosine deaminase.

Adenosine deaminase (ADA) deficiency causes severe combined immune deficiency (SCID) by interfering with the metabolism of deoxyadenosine, which is toxic to T lymphocytes at all stages of differentiation. Enzyme replacement with polyethylene glycol-modified ADA (PEG-ADA) has been previously shown to correct deoxyadenosine metabolism and improve mitogen-induced T lymphocyte proliferation. We studied the biochemical and immunologic effects of PEG-ADA in two infants with ADA-deficient SCID. While in a catabolic state, higher doses of PEG-ADA than previously described were required to normalize deoxyadenosine metabolism. After biochemical improvement, the patients recovered immune function in a pattern similar to that observed in normal thymic ontogeny and in patients with immunological reconstitution after bone marrow transplantation. Immune reconstitution was marked by the sequential appearance in the peripheral blood of phenotypic T lymphocytes corresponding to successive stages of thymic differentiation. Functional reconstitution was marked by the successive appearance of mitogen responses dependent on exogenous in vitro IL-2, mitogen responses not requiring exogenous IL-2, antigen-specific responses dependent on exogenous IL-2, and finally, antigen-specific responses not requiring exogenous IL-2. Natural killer function was tested in one patient and normalized with PEG-ADA therapy. Optimal PEG-ADA therapy appears to normalize thymic differentiation in ADA-deficient SCID, resulting in normal antigen-specific immune function.

Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients...

Is chronic graft versus host disease an autoimmune disease?

Chronic graft versus host disease continues to be a major problem following bone marrow transplantation even though the incidence and severity of acute graft versus host disease has been reduced. Recent investigations have suggested that the pathogenesis of chronic graft versus host disease is more similar clinically to an autoimmune disease than to acute graft versus host disease.

Severe combined immunodeficiency disease, adenosine deaminase deficiency and gene therapy.

Patients with severe combined immunodeficiency disease represent a model for the first clinical applications of gene therapy. Present attempts use insertion of the human adenosine deaminase gene into the peripheral blood T lymphocytes of patients who lack this gene. The ultimate treatment, however, will require insertion of the normal human adenosine deaminase gene into pluripotent stem cells and expression of the gene in their progeny.

In vitro purging of human rhabdomyosarcoma cells using 4-hydroperoxycyclophosphamide.

The outcome of patients with advanced stage rhabdomyosarcoma is extremely poor, with a disease-free survival of less than 20% at 3 years. Autologous bone marrow transplantation for patients with Clinical Group IV rhabdomyosarcoma may be an effective therapy. The bone marrow involvement diagnosed by light microscopy is 29% for patients with advanced disease. The present study was performed to test the ability of 4-hydroperoxycyclophosphamide (4-HC) to eliminate clonogenic rhabdomyosarcoma cells. Four different human rhabdomyosarcoma cell lines were treated in vitro with 4-HC at a concentration of 100 micrograms/ml. Limiting dilution analysis was performed to detect surviving clonogenic tumor cells. Treatment with 4-HC resulted in 1.7-5.7 log of elimination of clonogenic tumor cells in all four cell lines. Exactly the same log tumor cell kill was obtained after mixing normal human bone marrow mononuclear cells with rhabdomyosarcoma cells. Treatment with 4-HC may be an effective method of eliminating clonogenic rhabdomyosarcoma cells ex vivo.

Pharmacology studies of 1-beta-D-arabinofuranosylcytosine in pediatric patients with leukemia and lymphoma after a biochemically optimal regimen of loading bolus plus continuous infusion of the drug.

In an attempt to maximize the therapeutic index and to overcome the large variations in 1-beta-D-arabinofuranosylcytosine (ara-C) plasma levels and host toxicities that have been documented with standard HDara-C regimens (3 g/m2 over 3 h every 12 h x 8 or x12 doses), pediatric patients with acute lymphocytic leukemia or lymphoma in relapse were treated with a regimen of loading bolus followed immediately by continuous infusion of ara-C. In addition, patients received a single dose of etoposide (VP-16, 1 g/m2) prior to the ara-C administration. In four patients, total body irradiation was administered as part of a bone marrow transplantation preparative regimen after the ara-C administration. The regimen was designed to attain and maintain plasma steady-state concentrations (Css) of ara-C three to four times the Km2 value of ara-C, which was determined with purified deoxycytidine kinase from the patients' tumor cells prior to treatment. Eight patients age 0.75 to 16 years with relapsed acute lymphocytic leukemia (three patients) or lymphoma (five patients, one with bone marrow involvement), received a test dose of 3 g/m2 ara-C injected over 1 h, and the plasma kinetics were determined. The peak plasma ara-C concentration of ara-C ranged from 57 to 199 microM with an average concentration of 103 +/- 49 microM; the half-lives of distribution (t1/2, alpha) and elimination (t1/2, beta) averaged 17 +/- 7 min and 4.04 +/- 3.1 h, respectively. The mean area under the plasma concentration time curve from 0 to 12 h (AUC0----12 h) of ara-C averaged 386.8 +/- 328.0 microMh (mean, +/- SD, n = 8). The peak concentration of uracil arabinoside averaged 501 +/- 123 microM, and it was eliminated with a t1/2, el of 2.3 +/- 0.6 h. The patients then received an individualized loading bolus (mean = 0.5 g/m2) followed by a continuous infusion regimen of ara-C (mean = 130 mg/m2/h), to achieve a Css in the range of 20 to 35 microM. The obtained plasma Css were similar to the desired ones, averaging in variation 10.7% +/- 8.2%. The percentage of variation of correlation of the AUC following the loading bolus plus the continuous infusion from 12 to 72 h was only 12.4% (mean = 2158 microMh, n = 8), whereas the percentage of variation of correlation of the AUC after the test dose of ara-C in the same patients was 84.8%.(ABSTRACT TRUNCATED AT 400 WORDS)

Heparin inhibition of antibody-dependent complement-mediated lysis.

The effect of heparin on the monoclonal antibody-dependent complement-mediated lysis of human lymphoid cell lines and peripheral blood T-lymphocytes was studied. T-lymphoid cell lines (CEM and MOLT-3) were lysed by optimal concentrations of monoclonal antibodies and rabbit complement. Heparin at concentrations as low as 0.5 U/ml partially inhibited the complement-mediated lysis of all antibody-cell combinations while a heparin concentration of 25 U/ml produced complete inhibition. Lysis mediated by an IgG monoclonal antibody (J5) to the common acute lymphoblastic leukemia antigen (CALLA) was more sensitive to heparin inhibition than that due to an IgM antibody (VIL A1). Bovine and porcine heparin were equally inhibitory. Peripheral blood T-lymphocytes collected in heparin (100 U/ml) were resistant to complete lysis when treated with 17F12 and complement immediately after isolation; complete lysis was achieved only when they were incubated for 2 h prior to treatment. The role of monoclonal antibody and complement in the in vitro lysis of normal and leukemic lymphocytes is discussed.

Bone marrow transplantation for an infant with neutrophil dysfunction.

A child with severe neutrophil dysfunction and intractable infections received bone marrow transplants from histocompatible siblings. After a first transplant preceded by cyclophosphamide (CY), antithymocyte serum (ATS) and procarbazine (PCB) preconditioning, there was no evidence for engraftment and autologous marrow function rapidly returned. Cell mediated lysis showed no evidence of patient sensitization against the marrow donor suggesting that graft rejection did not cause the transplant failure. A second transplant was performed utilizing another matched sibling donor. Total body irradiation was added to CY, ATS, and PCB for preconditioning after in vitro studies of the colony forming capacity (CFUc) of the patient's marrow cells showed normal sensitivity to radiation. Full engraftment ensued with correction of granulocyte function abnormalities. The patient eventually died of intractable pulmonary disease. Our experience with this child suggests that cyclophosphamide alone may be insufficient preparation for marrow transplantation in some patients with non-neoplastic hematologic disorders. Experimental and clinical data supporting this contention are reviewed.

Human graft versus host disease.

Human graft versus host disease is composed of 2 distinct clinical entities, acute graft versus host disease and chronic graft versus host disease, which have different pathogenesis. Acute graft versus host disease is produced by the attack of donor immunocompetent T or null lymphocytes against recipient histocompatibility antigens. The null lymphocytes may attack antigens shared by the donor and recipient and are autocytotoxic lymphocytes which can produce acute graft versus host disease in recipients of identical twin transplants. The cessation of acute graft versus host disease occurs when suppressor lymphocytes appear in the recipient's peripheral circulation. Chronic graft versus host disease is produced by immunocompetent lymphocytes that differentiate in the recipient. Its control is unknown. Some patients with chronic graft versus host disease have in vivo activated suppressor lymphocytes which produce a secondary immunoincompetence and an increased susceptibility to bacterial sepsis and death.