Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z⁺ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1⁺ or 19z1-CD80⁺ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z⁺ T cells had 10-fold reduced tumor progression compared to those treated with 19z1⁺ or 19z1-CD80⁺ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80⁺ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z⁺ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.     

The quest for spatio-temporal control of CAR T cells

Chimeric antigen receptors (CARs) are synthetic receptors capable of directing potent antigen-specific anti-tumor T cell responses. A recent report by Wu et al. extends a series of strategies aiming to curb excessive T cell activity, utilizing in this instance a chemical dimerizer to aggregate antigen-binding, T cell-activating and costimulatory domains.Chimeric antigen receptor (CAR) therapy relies on T cell engineering to generate tumor-targeted T cells with enhanced anti-tumor functions¹. CAR therapy has so far achieved its most remarkable clinical successes against CD19-positive hematological malignancies and is now on the verge of being developed for solid tumors². Two safety concerns have, however, emerged from the CD19 experience, which should be addressed for CAR therapy to be broadly applicable. One is the eventual on-target/off-tumor effect of CAR T cells on normal tissues. Even though this concern may be mitigated in the case of CD19 CAR T cell-induced B cell aplasia, strategies designed to reduce or prevent its potential occurrence with other targets are needed². The other concern is a severe cytokine release syndrome (CRS), arising from large-scale synchronized T cell activation upon engaging the target antigen in some CAR T cell recipients².Several innovative strategies have been recently proposed to address these safety concerns. These strategies make use of remote or cell autonomous controls (Figure 1), utilizing small molecules, antibodies or synthetic receptors to regulate T cell activity. One approach is to activate a latent suicide switch, such as the inducible caspase-9 (iCasp9) enzyme, through the administration of a small molecule to induce T cell apoptosis³ (Figure 1a). Bifunctional small molecules that mediate the binding between antigen and CAR have also been developed to regulate target engagement⁴ (Figure 1b). A variation on this approach uses antibodies to mediate antigen recognition on target cells and binding of T cells expressing a synthetic Fc receptor⁵ (Figure 1b). These designs enable remote temporal control of T cell activity but do not provide a means to enhance tumor selectivity of the CAR T cells. To this end, combinatorial approaches integrating two autonomous antigen inputs to control CAR T cell functions have been developed to spatially discriminate between normal and tumor cells expressing a common target. One such approach utilizes synthetic inhibitory receptors, termed iCARs, which are derived from the PD-1 or CTLA-4 receptors, to protect normal cells based on the iCAR''s recognition of an antigen present on the normal cells but not the tumor cells⁶ (Figure 1c). Another approach utilizes complementary signals split between two receptors — a CAR for T cell activation and a chimeric costimulatory receptor (CCR) providing costimulation — such that they are both expressed by the tumor cells but found alone on normal cells⁷ (Figure 1d). Acting in cell autonomous fashion, the required co-engagement of the CCR and the CAR upon recognition of two independent antigens reinforces tumor selectivity in vivo⁷.Open in a separate windowFigure 1Building controls into engineered T cells. (a) The small molecule AP1903 can dimerize the suicide switch iCasp9 to induce T cell apoptosis. (b) Bifunctional small molecule bridging the binding between antigen and CAR or antibody mediating the interaction between antigen and synthetic Fc receptor can be remote controls of CAR T cells. (c) iCAR can inhibit CAR function in the presence of an antigen present in normal cells but not tumor cells. (d) CCR binding to a second antigen in tumor cells is required for full T cell activation. (e) The small molecule AP21976 can dimerize two independent signaling entities through an FKBP-FRB module to control T cell activation. (a, b, e) Strategies employing one remote control (antibody or small molecule) in addition to one autonomous control (antigen A). (c, d) Strategies with two autonomous controls (antigen A and antigen B). Negative regulation involves inducing apoptosis (a) or turning off T cell activation (c) by input 2 while positive regulation (b, d, e) results in T cell activation by input 2.In a recent paper published in Science, Wu et al.⁸ showed a novel design incorporating a remote control of CAR T cells, whereby a small molecule is used to dimerize antigen-binding and signaling domains (Figure 1e). At variance with the small molecule-controlled suicide switch, this ON-switch design represents a positive reversible regulation, as it does not eliminate T cells but rather restricts their activities. The remote control takes advantage of well-established chemically induced dimerization (CID) modules developed in the 1990s, where two proteins bind only in the presence of a third chemical, such as a small molecule⁹. One such widely used CID module is the FKBP and FRB_T2098L that heterodimerize in the presence of rapamycin or its less immunosuppressive analog AP21976. The receptor for antigen and a dual-signaling, costimulatory and activating domain analogous to that of a second generation CAR, were independently fused to FKBP and FRB_T2098L so that AP21976-induced FKBP and FRB_T2098L dimerization could aggregate these entities (Figure 1e). This design controls intracellular assembly of a signaling complex without affecting the antigen binding properties as afforded by the bifunctional small molecules or antibodies at the interface of T cells and target cells (Figure 1b). After screening various domain configurations in leukemic Jurkat cells with AP21976-dependent NFAT activation and IL-2 production assays, a design that worked with both the FKBP-FRB_T2098L and the gibberellin-induced GID1-GAI heterodimerization modules was identified. Single molecule imaging of ON-switch CAR assembly in Jurkat cells showed that two molecular parts are equally constrained to immobilized antigens only in the presence of AP21976. Subsequent characterization of the ON-switch CAR in primary human CD4⁺ T cells showed that both AP21976 and antigen are required for the induction of CD69 expression, a biomarker of T cell activation, the secretion of both IL-2 and IFNγ, and the proliferation of CD4⁺ cells. Most gratifyingly, there was a positive correlation between these responses and the AP21976 dosage, suggesting the possibility of achieving titratable control of T cells. Human primary CD8⁺ T cells with ON-switch CAR in three different cytotoxicity assays also demonstrated antigen- and AP21976-dependent killing of tumor cells, which was also titratable by AP21976. The killing ability of ON-switch CAR CD8⁺ T cells was reversible, as removal of AP21976 abrogated tumor cell lysis.Wu et al. proceeded to explore in vivo activity in a mouse xenograft model. Due to the short plasma half-life and the high cost of AP21976, the study is limited to a very short-term protocol of 39 h. Tumor cells were injected into the peritoneal cavity 14 h prior to the injection of the engineered T cells. Four injections of AP21976 in the subsequent 25 h were required to induce anti-tumor activity in this intraperitoneal cytotoxicity assay. Further investigations with a more relevant protocol allowing for tumor engraftment and longer term follow-up of T cell effectiveness will be needed to establish whether AP21976 can remotely control ON-switch CAR T cells to reject a tumor.Wu and coauthors have thus engineered a novel ON-switch CAR design and demonstrated titratable, reversible and antigen-dependent T cell functions controlled by a dimerizing small molecule. Another group is also conducting preclinical studies exploring a variant small molecule-controlled CAR design for solid tumor rejection¹⁰. However, there are still challenges to address before future clinical applications. The authors pointed out the need to develop controller chemicals that have clinically optimized pharmacokinetic properties, as the half-life of AP21976 is short and impractical for clinical application. Thus, how many injections per day, for how many weeks or months, would be required to achieve tumor rejection? Another unresolved question is whether a small molecule with optimal pharmacokinetic properties could effectively curb CRS and off-tumor reactivity. Overall, this elegant study provides valuable insights for further refining spatio-temporal control of cell therapy and applying it to CAR T cell technology.     

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell-derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell-derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell-derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.     

Ligand binding to inhibitory killer cell Ig-like receptors induce colocalization with Src homology domain 2-containing protein tyrosine phosphatase 1 and interruption of ongoing activation signals

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.     

The potential of stem cells as an in vitro source of red blood cells for transfusion

Recent advances have increased excitement about the potential for therapeutic production of red blood cells (RBCs) in vitro. However, generation of RBCs in the large numbers required for transfusion remains a significant challenge. In this article, we summarize recent progress in producing RBCs from various cell sources, and discuss the hurdles that remain for translation into the clinical arena.     

Frequency of Missense Mutations in the Coding Region of a Eukaryotic Gene Transferred by Retroviral Vectors

A relatively high mutation rate is probably a major factor in the evolutionary success of retroviruses, because it generates the genetic diversity that helps them to cope with changes in the environment. When using recombinant retroviruses as vectors for gene transfer and gene therapy, it is important to consider the implications of this biological characteristic. Until now, the mutation rate has been studied by using noneukaryotic genes as reporters. Here we report point mutations in the human glucose-6-phosphate dehydrogenase (hG6PD) gene transferred by Moloney murine leukemia virus-based vectors into murine bone marrow cells and NIH 3T3 murine fibroblasts. After bone marrow transplantation, we observed an hG6PD with abnormal electrophoretic mobility for 2 out of 34 mice. Next, we studied this phenomenon quantitatively and found 1 electrophoretically abnormal hG6PD variant among 93 independently isolated NIH 3T3 clones, from which we estimate a mutation rate of 1.4 x 10(-5) per base pair per replication cycle. Mutations in the transferred gene can thus contribute to the impairment of the effectiveness of retrovirus-mediated gene transfer.     

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.