Cell Density Plays a Critical Role in Ex Vivo Expansion of T Cells for Adoptive Immunotherapy

The successful ex vivo expansion of a large numbers of T cells is a prerequisite for adoptive immunotherapy. In this study, we found that cell density had important effects on the process of expansion of T cells in vitro. Resting T cells were activated to expand at high cell density but failed to be activated at low cell density. Activated T cells (ATCs) expanded rapidly at high cell density but underwent apoptosis at low cell density. Our studies indicated that low-cell-density related ATC death is mediated by oxidative stress. Antioxidants N-acetylcysteine, catalase, and albumin suppressed elevated reactive oxygen species (ROS) levels in low-density cultures and protected ATCs from apoptosis. The viability of ATCs at low density was preserved by conditioned medium from high-density cultures of ATCs in which the autocrine survival factor was identified as catalase. We also found that costimulatory signal CD28 increases T cell activation at lower cell density, paralleled by an increase in catalase secretion. Our findings highlight the importance of cell density in T cell activation, proliferation, survival and apoptosis and support the importance of maintaining T cells at high density for their successful expansion in vitro

Anti-KIT designer T cells for the treatment of gastrointestinal stromal tumor

Background: Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells. Methods: Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions. Results: We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates. Conclusions: We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc

Solar Neutrino Constraints on the BBN Production of Li

Using the recent WMAP determination of the baryon-to-photon ratio, 10^{10} \eta = 6.14 to within a few percent, big bang nucleosynthesis (BBN) calculations can make relatively accurate predictions of the abundances of the light element isotopes which can be tested against observational abundance determinations. At this value of \eta, the Li7 abundance is predicted to be significantly higher than that observed in low metallicity halo dwarf stars. Among the possible resolutions to this discrepancy are 1) Li7 depletion in the atmosphere of stars; 2) systematic errors originating from the choice of stellar parameters - most notably the surface temperature; and 3) systematic errors in the nuclear cross sections used in the nucleosynthesis calculations. Here, we explore the last possibility, and focus on possible systematic errors in the He3(\alpha,\gamma)Be7 reaction, which is the only important Li7 production channel in BBN. The absolute value of the cross section for this key reaction is known relatively poorly both experimentally and theoretically. The agreement between the standard solar model and solar neutrino data thus provides additional constraints on variations in the cross section (S_{34}). Using the standard solar model of Bahcall, and recent solar neutrino data, we can exclude systematic S_{34} variations of the magnitude needed to resolve the BBN Li7 problem at > 95% CL. Additional laboratory data on He3(\alpha,\gamma)Be7 will sharpen our understanding of both BBN and solar neutrinos, particularly if care is taken in determining the absolute cross section and its uncertainties. Nevertheless, it already seems that this ``nuclear fix'' to the Li7 BBN problem is unlikely; other possible solutions are briefly discussed.Comment: 21 pages, 3 ps figure

High-dose intravenous gamma globulin to suppress alloimmune destruction of donor platelets

We report the use of high-dose intravenous gamma globulin to overcome refractoriness to platelet transfusion in an alloimmunized patient with acute leukemia and thrombocytopenia. For two years the patient suffered recurrent gastrointestinal bleeding from an arteriovenous malformation and was given multiple transfusions, providing a basis for his allosensitization. Platelet counts had not increased following transfusions of random-donor or HLA-matched platelets. With intravenous gamma globulin, one hour after the transfusion of 9 to 15 units of platelets, the count increased by 30,000 to 90,000 and the half-life of transfused platelets increased to three to four hours from an estimated 0.05 hours prior to therapy. Intravenous gamma globulin arrested massive gastrointestinal bleeding and allowed the patient to undergo surgical resection of the small bowel with minimal operative blood loss

YY1 Upregulates Checkpoint Receptors and Downregulates Type I Cytokines in Exhausted, Chronically Stimulated Human T Cells

Summary: T cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-γ). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28→p38MAPK/JNK→YY1→ exhaustion. : Cancer Systems Biology; Immune Response; Immune System Disorder Subject Areas: Cancer Systems Biology, Immune Response, Immune System Disorde

Recombination–deletion between homologous cassettes in retrovirus is suppressed via a strategy of degenerate codon substitution

Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination–deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination–deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence