Attenuation of CD4+ T-cell function by human adenovirus type 35 is mediated by the knob protein.

The complement-regulatory protein CD46 is the primary receptor for human adenovirus type 35 (HAdV-35) and can regulate human immune-cell activation. CD4(+) T-cells are critical for initiating and maintaining adaptive immunity elicited by infection or vaccination. It was reported previously that HAdV-35 can bind these cells and suppress their activation. The data reported here demonstrate that recombinant trimeric HAdV-35 knob proteins alone can induce CD46 receptor downregulation and inhibit interleukin-2 production and proliferation of human CD4(+) T-cells in vitro similarly to mAbs specific to the CD46 region bound by HAdV-35 knobs. A mutant knob protein with increased affinity for CD46 compared with the wild-type knob caused equivalent effects. In contrast, a CD46-binding-deficient mutant knob protein did not inhibit T-cell activation. Thus, the capacity of HAdV-35 to attenuate human CD4(+) T-cell activation depends predominantly on knob interactions with CD46 and can occur independently of infection.

Evaluation of safety and pharmacokinetics of sodium 2,2 dimethylbutyrate, a novel short chain fatty acid derivative, in a phase 1, double-blind, placebo-controlled, single-dose, and repeat-dose studies in healthy volunteers.

Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease (SCD) and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate (AB), and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous (IV) infusion. A fetal globin-inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases. Healthy adult human subjects were treated with a novel short chain fatty acids (SCFA) derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with 1 of 4 single dose levels (2, 5, 10, and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticulocytes, and HbF assays. SDMB was well-tolerated with no clinically significant adverse events related to study medication. The terminal half-life ranged from 9 to 15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14-day study. The favorable pharmacokinetics (PK) profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies.

Fetal globin gene inducers: novel agents and new potential.

Inducing expression of endogenous fetal globin (gamma-globin) gene expression to 60-70% of alpha globin synthesis produces beta-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced gamma-globin expression in beta-thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as seven years. However, prior generation inducers were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic, sodium 2,2-dimethylbutyrate, is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of three major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used worldwide.

Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis.

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Factors affecting human T cell engraftment, trafficking, and associated xenogeneic graft-vs-host disease in NOD/SCID beta2mnull mice.

OBJECTIVE: Graft-vs-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Models of immunodeficient mice that consistently and efficiently reconstitute with xenoreactive human T cells would be a valuable tool for the in vivo study of GVHD, as well as other human immune responses. MATERIALS AND METHODS: We developed a consistent and sensitive model of human GVHD by retro-orbitally injecting purified human T cells into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID)-beta2m(null) recipients. In addition, we characterized for the first time the trafficking patterns and expansion profiles of xenoreactive human T cells in NOD/SCID-beta2m(null) recipients using in vivo bioluminescence imaging. RESULTS: All NOD/SCID-beta2m(null) mice conditioned with 300 cGy total body irradiation and injected with 1 x 10(7) human T cells exhibited human T-cell engraftment, activation, and expansion, with infiltration of multiple target tissues and a subsequent >20% loss of pretransplantation body weight. Importantly, histological examination of the GVHD target tissues revealed changes consistent with human GVHD. Furthermore, we also showed by in vivo bioluminescence imaging that development of lethal GVHD in the NOD/SCID-beta2m(null) recipients was dependent upon the initial retention and early expansion of human T cells in the retro-orbital sinus cavity. CONCLUSION: Our NOD/SCID-beta2m(null) mouse model provides a system to study the pathophysiology of acute GVHD induced by human T cells and aids in development of more effective therapies for human GVHD.

In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of leukemic B cells concomitant with immunological abnormalities and depressed immune responses. The T cell abnormalities found in CLL patients are thought to increase the risk of infection and hamper immune recognition and elimination of leukemic cells. We evaluated whether providing signals through CD3 and CD28 would correct some of these T cell defects. PBMC were incubated with anti-CD3 and anti-CD28 mAbs conjugated to superparamagnetic beads for 12-14 days. This resulted in a 1400-fold increase in T cell numbers. Activated T cells expressed high levels of CD25, CD54, CD137, and CD154, and produced IFN-gamma, TNF-alpha, and GM-CSF. The mean T cell composition of cultures increased from approximately 6% to >90% and leukemic B cells decreased from a mean of approximately 85% to 0.1% or less. Leukemic B cells up-regulated expression of CD54, CD80, CD86, and CD95. Receptor up-regulation required direct cell contact with the activated T cells and could be blocked with anti-CD154 mAb, suggesting that the CD40-CD40L pathway helped mediate these effects. Poor T cell responses to allostimulation were corrected by the activation and expansion process. The skewing in the TCR repertoire returned to normal, or near normal following the culture process in eight of nine patients with abnormal TCR repertoires. Activated T cells had potent in vitro antileukemic effects in contrast to nonactivated T cells. Based upon these findings, a clinical trial has been initiated to test the potential therapeutic effects of T cells activated using this approach in patients with CLL.

Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies.

T-cell receptor engagement and accompanying costimulatory signals control the level of activation and functional potential of individual T cells. The authors previously developed a novel technology in which human T cells are activated and expanded in culture ex vivo using anti-CD3 and anti-CD28 monoclonal antibodies covalently linked to superparamagnetic beads (Xcyte Dynabeads). In this study the addition of N-acetyl L-cysteine (NAC) to the cultures markedly increased the expansion of T cells from human peripheral blood mononuclear cells without diminishing cell function. NAC increased the rate of T-cell division, reduced apoptosis, and increased the percentage of antigen-specific memory T cells in the cultures. The effect of varying the ratio of beads to T cells (1:10-10:1) at culture initiation was also evaluated. Polyclonal T cells were expanded at all bead-to-T cell ratios tested (range 1:10-10:1). While high bead-to-T cell ratios (5:1 and 10:1) deleted, low ratios (1:10 and 1:5) preserved memory T cells directed against cytomegalovirus, Epstein-Barr virus, and influenza virus antigens. Adding more anti-CD3/anti-CD28 beads during the culture led to further expansion of T cells. Experiments also revealed that reducing the amount of anti-CD3 antibodies relative to the amount of anti-CD28 antibodies on the beads favored the proliferation of antigen-specific T cells. In summary, these data indicate that T cell-stimulating effects of anti-CD3/anti-CD28 beads can be further manipulated to control the expansion of antigen-specific memory T cells and can be used to rapidly expand antigen-specific T cells ex vivo for potential clinical applications.

A phase I trial of CD3/CD28-activated T cells (Xcellerated T cells) and interleukin-2 in patients with metastatic renal cell carcinoma.

PURPOSE: Xcellerated T Cells (Xcyte Therapies, Seattle, WA) are autologous T cells that have been activated and expanded ex vivo using antibodies to CD3 and CD28 coimmobilized on magnetic beads. This study assessed the safety, immunostimulatory effects, and antitumor activity of Xcellerated T Cells and interleukin-2 (IL-2) in patients with metastatic renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: Twenty-six patients with measurable metastatic RCC after prior nephrectomy underwent leukapheresis. Peripheral blood lymphocytes were stimulated ex vivo for 8 days. Two cycles of therapy separated by 28 days were planned, with each cycle consisting of i.v. Xcellerated T Cells on day 1 and s.c. IL-2 at 10 x 10(6) units on days 1-10. RESULTS: Forty-nine cycles of therapy were administered to 25 patients. A mean (+/-SD) of 21.8 x 10(9) (+/-5.4 x 10(9)) Xcellerated T Cells were administered each cycle. The infused cells were 94% +/- 3% CD3(+), 64 +/- 12% CD4(+), and 25 +/- 12% CD8(+) (mean +/- SD). Adverse events (most commonly, fever and an influenza-like syndrome) were mild to moderate. Two patients developed significantly elevated human antimouse antibody titers (HAMA). No complete or partial clinical responses were observed. However, two patients experienced significant tumor regression in bone metastases. Median survival was 21 months. The number of cells infused correlated with the peak absolute lymphocyte count achieved, and there was a trend to increased postinfusion survival in patients achieving higher peak absolute lymphocyte counts. CONCLUSIONS: Adoptive immunotherapy with Xcellerated T Cells and IL-2 can be carried out safely on an outpatient basis in patients with advanced RCC. Further investigation of this therapy is warranted.