Showing papers by "David Baltimore published in 2018"

Functional TCR T cell screening using single-cell droplet microfluidics

Abstract: Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.

Isolation and characterization of NY-ESO-1–specific T cell receptors restricted on various MHC molecules

Abstract: Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2–restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable

Abstract: HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs) We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors

Author Correction: An NF-κB-microRNA regulatory network tunes macrophage inflammatory responses.

Abstract: Li-Fan Lu and Alexander Y. Rudensky, who supplied miR-146a floxed mice used in this study, were inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article. The generation of the floxed mice has been described in detail by Cho and Lee et al.1.

Dendritic cells efficiently transmit HIV to T Cells in a tenofovir and raltegravir insensitive manner

Abstract: Dendritic cell (DC)-to-T cell transmission is an example of infection in trans, in which the cell transmitting the virus is itself uninfected. During this mode of DC-to-T cell transmission, uninfected DCs concentrate infectious virions, contact T cells and transmit these virions to target cells. Here, we investigated the efficiency of DC-to-T cell transmission on the number of cells infected and the sensitivity of this type of transmission to the antiretroviral drugs tenofovir (TFV) and raltegravir (RAL). We observed activated monocyte-derived and myeloid DCs amplified T cell infection, which resulted in drug insensitivity. This drug insensitivity was dependent on cell-to-cell contact and ratio of DCs to T cells in coculture. DC-mediated amplification of HIV-1 infection was efficient regardless of virus tropism or origin. The DC-to-T cell transmission of the T/F strain CH077.t/2627 was relatively insensitive to TFV compared to DC-free T cell infection. The input of virus modulated the drug sensitivity of DC-to-T cell infection, but not T cell infection by cell-free virus. At high viral inputs, DC-to-T cell transmission reduced the sensitivity of infection to TFV. Transmission of HIV by DCs in trans may have important implications for viral persistence in vivo in environments, where residual replication may persist in the face of antiretroviral therapy.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

Abstract: For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell-T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

Bud13 Promotes a Type I Interferon Response by Countering Intron Retention in Irf7

Abstract: Intron retention (IR) occurs in the transcripts of certain genes involved in the inflammatory response. IR decreases the amount of mRNA that can be produced from a given amount of transcript and therefore reduces the protein output from the gene. IR occurs at one intron of the Irf7 gene, an important transcription factor for producing interferons. We have identified Bud13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in Bud13 leads to increased IR, decreased mature Irf7 transcript and protein levels, and consequently to a dampened type I interferon response. Viral infection of Bud13-deficient cells revealed that the impairment of Irf7 production compromises their ability to withstand VSV infection. Global analysis of Bud13 knockdown and BUD13 cross-linking to RNA reveals that a subset of introns, with similar characteristics to the one found in Irf7 (short, GC-rich with poor splice donor sequences), are spliced in a Bud13-dependent manner, increasing mRNA production from these genes. Thus, by acting as an antagonist to IR, Bud13 upregulates the expression of genes at which IR occurs.

Programmed Delayed Splicing: A Mechanism for Timed Inflammatory Gene Expression

Abstract: Inflammation involves timed gene expression, suggesting that the fine-tuned onset, amplitude, and termination of expression of hundreds of genes is of critical importance to organismal homeostasis. Recent study of post-transcriptional regulation of inflammatory gene expression led to the suggestion of a regulatory role for pre-mRNA splicing. Here, using a hybrid capture approach to purify incompletely spliced, chromatin-associated pre-mRNAs, we use deep sequencing to study pre-mRNA splicing of the NF-kB transcriptome. By freezing transcription and examining subsequent splicing of complete transcripts, we find many introns splice tens to hundreds of times slower than average. In many cases, this is attributable to poor splice donor sequences that are evolutionarily conserved. When these introns were altered by ~2 base pairs to yield stronger splice donors, gene expression levels increased markedly for several genes in the context of a reporter system. We propose that such splice sites represent a regulatory mechanism that determines the timing of production of the mRNAs from certain inflammatory genes and may also limit mRNA expression from these genes. Further work will be needed to understand the roles of this regulation in the inflammatory response. The suggestion of extensive temporal regulation of pre-mRNA splicing as a regulatory process in inflammation raises the question of where else in biology there may be timed processes with a similar underlying cause.

Sensitive, Non‐Destructive Detection and Analysis of Neoantigen‐Specific T Cell Populations from Tumors and Blood

Abstract: Tumor neoantigens are fragments of mutated proteins that contain the mutation, and can be presented by major histocompatibility complex molecules on tumor cells, where they are surveyed by T cells. The rapid and sensitive identification of neoantigen‐specific T cell populations from tumor tissues or blood has proven challenging. A microchip platform for the non‐destructive identification of neoantigen‐specific CD8 T cells is described. The method utilizes a library of neoantigen/MHC tetramers linked to a magnetic nanoparticle via a DNA barcode. The neoantigen‐specificity of the T cells is determined by decoding the barcode through sequential fluorescent microscopy reads. The captured T cells may be further characterized for function, or via matching the neoantigen‐specificity with the T cell receptor gene. Tumor infiltrating lymphocytes and non‐expanded peripheral blood mononuclear cells collected from melanoma patients at various time points across an anti‐PD1 therapy regimen are shown to contain overlapping neoantigen‐specific T cell populations.

Signaling and antigen-presenting bifunctional receptors (sabr)

Abstract: Described herein are compositions and methods for signaling and antigen-presenting bifunctional receptors (SABRs) comprising one or more antigen presenting domains; and one or more signal transduction domains, wherein the one or more antigen presenting domains comprise a binding fragment of a major histocompatibility complex (MHC) molecule. Various immunological functions of the SABRs are also described.

Characterization of Postinfusion Phenotypic Differences in Fresh Versus Cryopreserved TCR Engineered Adoptive Cell Therapy Products.

Abstract: Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.

Bridging the Science-Law Divide

Abstract: Formal opportunities for members of the scientific and legal communities to engage in ongoing collegial consideration of issues at the interface of science and law are limited. In the late 1990s, t...

Trogocytosis mediated epitope discovery

Abstract: Disclosed herein are trogocytosis based TCR ligand discovery platforms and methods of using the same, trogocytosis based TCR discovery platforms and methods of using the same, and trogocytosis based ligand discovery platforms and methods of using the same. Also disclosed herein are isolated cells, nucleotides, sequences, and sequence databases produced using trogocytosis based discovery platforms.