Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers.

Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allogeneic stem cell transplantation is a cause of morbidity and mortality from viral pneumonitis. Antiviral drugs given to reactivating patients have reduced the mortality from CMV but have toxic side effects and do not always prevent late CMV disease. Cellular immunotherapy to prevent CMV disease is less toxic and could provide prolonged protection. However, a practical approach to generating sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is required. This study describes a system for generating sufficient CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 bone marrow transplant recipients from 200 mL donor blood. Donor monocytes are used to generate dendritic cells (DCs) in medium with autologous plasma, interleukin 4, granulocyte-macrophage colony-stimulating factor, and CD40 ligand. The DCs are pulsed with the immunodominant HLA-A*0201-restricted CMV peptide pp65(495-503), and incubated with donor T cells. These cultures are restimulated twice with peptide-pulsed lymphoblastoid cell lines (LCLs) or CD40-ligated B cells and purified with phycoerythrin (PE)-labeled pp65(495-503)/HLA-A*0201 tetramers by flow sorting, or with anti-PE paramagnetic beads. The pure tetramer-positive population is then rapidly expanded to obtain sufficient cells for clinical immunotherapy. The expanded CTLs are more than 80% pure, of memory phenotype, with a Tc1 cytokine profile. They efficiently kill CMV-infected fibroblasts and express the integrin VLA-4, suggesting that the CTLs could cross endothelial barriers. This technique is reproducible and could be used for generating CMV-specific CTLs to prevent CMV disease after allogeneic blood and marrow transplantation. (Blood. 2001;98:505-512)

Detection of xenoantibodies using a simple flow cytometric assay.

Higher primates, including humans, have high levels of pre-existing naturally circulating antibodies that predominantly recognize the epitope Gal (1,3-Gal), which is highly expressed on the surface of xenogenic cells. Deposition of these antibodies on the endothelial cell surface of vascularized xenografts leads to an activation of the classical pathway of the complement system, resulting in tissue ischemia and necrosis with rapid demise of the xenograft. This hyperacute rejection (HAR) is always a major barrier in xenograft transplantation and should be minimized by accurately monitoring the naturally occurring antibodies. In the present study, we utilized a simple and rapid flow cytometric (FCM) assay to monitor the presence of these naturally occurring antibodies. We found that the FCM assay is very effective in measuring human antibodies bound to the xenogenic cells, which cause cytotoxicity. This assay could be useful in the pre- and post-xenotransplantation monitoring of xenoantibodies, thus, helping in the development of strategies to block the binding of preformed human antibodies to the xenograft in order to overcome the problem of HAR.

Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats.

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 single-density, or by 1.077/1.119 double-density centrifugation, the 1.077/1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously.

The safety and activity of polymerized ragweed: a double-blind, placebo-controlled trial in 81 patients with ragweed rhinitis.

Eighty-one patients with ragweed pollinosis were recruited for a double-blind, histamine placebo-controlled study of the safety, immunogenicity, and efficacy of 15 weekly injections of polymerized ragweed (PRW) immunotherapy totaling 1359 allergy units. Patients were paired on the basis of cutaneous end point titration to RAST standardized extracts of giant and short ragweed. One patient of each pair was randomized to receive PRW, and the other patient, a caramelized glucose histamine placebo. Symptom and medication score diaries were completed by 68 patients. All 68 patients received the full maintenance dose. No patient dropped out because of adverse reactions, and there were no systemic reactions. Except for one patient receiving placebo who developed mildly elevated liver function tests, there were no clinically significant changes in routine laboratory tests associated with injections. By Student's test on log-transformed values, blocking antibody rose significantly in the patients receiving PRW but was unchanged in those receiving placebo. By Wilcoxon paired signed-rank test, the symptom and medication scores in the patients receiving PRW were significantly lower than scores in the patients receiving placebo. This study demonstrates the safety, immunogenicity, and activity of PRW in the treatment of ragweed pollinosis.

Immunologic studies in schizophrenic and control subjects.

Heath and coworkers proposed that schizophrenia may be an autoimmune disorder in which antibodies are built up against specific substances in certain brain cells. Heath reports that schizophrenic patients exhibit abnormal brain waves in recordings from the caudate nucleus and septal area. These abnormal waves can also be recorded from similar sites in monkey brains after injections into the lateral ventricle cerebrospinal fluid of gamma-G-immunoglobulins (IgG) isolated from the blood of acutely ill schizophrenic patients. We prepared IgG fractions from control subjects and acutely ill schizophrenic patients and tested them in rhesus monkeys under double-blind conditions. Of 107 sera tested from 24 schizophrenic patients, 29 produced positive electroencephalographic recordings in the monkeys. From 30 control subjects we tested 80 samples and found 6 to be positive according to Heath's criteria. This amounts to more than 1 positive reaction for every 4 schizophrenic patients' fractions tested and approximately 1 positive in 13 from control subjects' serum fractions. The difference between control and patient groups is highly significant (p less than 0.001). Although our results confirm the experimental findings of the Heath group concerning abnormal EEG activity associated with an IgG fraction from schizophrenic patients, they differ from Heath's results for fractions from control persons. We found positive effects from a small number of control fractions whereas Heath claims never to have observed positive biological activity in control fractions. The autoimmune hypothesis has numerous drawbacks, the greatest of which is the inability to demonstrate the presence of circulating antibody in schizophrenic patients with the use of standard immunologic techniques.