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Chimeric antigen receptors (CAR) combine an antigenbinding domain with a CD3-Z signaling motif to redirect Tcell specificity to clinically important targets. First-generation CAR, such as the CD19-specific CAR (designated CD19R), may fail to fully engage genetically modified T cells because activation is initiated by antigen-dependent signaling through chimeric CD3-Z, independent of costimulation through accessory molecules. We show that enforced expression of the fulllength costimulatory molecule CD28 in CD8 + CD19R + CD28À T cells can restore fully competent antigen-dependent T-cell activation upon binding CD19 + targets expressing CD80/CD86. Thus, to provide costimulation to T cells through a CD19-specific CAR, independent of binding to CD80/CD86, we developed a second-generation CAR (designated CD19RCD28), which includes a modified chimeric CD28 signaling domain fused to chimeric CD3-Z. CD19R + and CD19RCD28 + CD8 + T cells specifically lyse CD19 + tumor cells. However, the CD19RCD28 + CD8 + T cells proliferate in absence of exogenous recombinant human interleukin-2, produce interleukin-2, propagate, and up-regulate antiapoptotic Bcl-X L after stimulation by CD19 + tumor cells. For the first time, we show in vivo that adoptively transferred CD19RCD28 + T cells show an improved persistence and antitumor effect compared with CD19R + T cells. These data imply that modifications to the CAR can result in improved therapeutic potential of CD19-specific T cells expressing this second-generation CAR. (Cancer Res 2006; 66(22): 10995-1004)

Conceptualizing the households of working-class Latino students as being rich in funds of knowledge has had transformative consequences for teachers, parents, students, and researchers. Teachers' qualitative, ethnographic study of their own students' households has unfolded as a viable method for bridging the gap between school and community. The focus of the home visit is to gather details about the accumulated knowledge base that each household assembles in order to ensure its own subsistence. Teachers also participate in study groups that offer a forum for the collective analysis of the household findings, and they form curriculum units that tap into the household funds of knowledge. New avenues of communication between school and home foster confianza, or mutual trust.

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Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells. (J. Clin. Invest. 1994. 94:797-807.)

What can be learned from the Puente experience about identifying and incorporating local funds of knowledge of Latino communities into precollege preparation? This article focuses on how Puente teachers and students can enhance their practice and mutual learning through ethnographic fieldwork in the students' home community. Through investigating the many local funds of knowledge that can be utilized to validate students' identities as knowledgeable individuals who can use such knowledge as a foundation for future learning, both teachers and students can engage in a critical pedagogy predicated on resources and not deficits.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

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