Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1.

A major challenge in developing an effective adoptive cancer immunotherapy is the ex-vivo generation of tumor-reactive cells in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases their cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL) from melanoma patients, following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate and a lower frequency of TIM-3+CD8+ cells after stimulation with anti-CD3/CD28 beads. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that included a 14-day rapid expansion procedure (REP). Co-incubation of post-REP TIL with matching target cancerous cells demonstrated increased IFNγ secretion beyond baseline in most of the TIL cultures, as well as a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25, several exhaustion markers and the differentiation status of the expanded cells. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy for cancer patients.

Tumor-infiltrating lymphocytes from human prostate tumors reveal anti-tumor reactivity and potential for adoptive cell therapy.

Advanced prostate cancer remains incurable and is the second leading cause of mortality in men. Immunotherapy based on the adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical results in patients with metastatic melanoma and lately also in other solid tumors. However, the ability to obtain TIL from patients with prostate cancer, considered poorly immunogenic, remains unknown. In this study, we investigate the feasibility of isolating and expanding TIL from primary prostate tumors. We collected tumor specimens from eight patients with diagnosed prostate adenocarcinoma undergoing radical prostatectomy and were able to successfully expand multiple autologous TIL cultures from all patients. Twenty-eight prostate-TIL cultures were further expanded using a standard rapid expansion procedure under Good Manufacturing Practice conditions. TIL cultures were phenotypically characterized for T cell subset composition, differentiation status and co-inhibitory/stimulatory markers such as PD-1, TIM-3, LAG-3, and CD28 and were found to have in general similarity to TIL obtained from patients with melanoma and lung carcinoma previously treated at our center. All analyzed TIL cultures were functional as determined by the capability to produce high level of IFNγ upon stimuli. Most importantly, co-culture assays of prostate-TIL with autologous tumors demonstrated anti-tumor reactivity. In conclusion, these findings demonstrate that functional and anti-tumor reactive TIL can be obtained, despite the immunosuppressive microenvironment of the cancer, thus this study supports the development of TIL therapy for prostate cancer patients.

Level of neo-epitope predecessor and mutation type determine T cell activation of MHC binding peptides.

BACKGROUND: Targeting epitopes derived from neo-antigens (or "neo-epitopes") represents a promising immunotherapy approach with limited off-target effects. However, most peptides predicted using MHC binding prediction algorithms do not induce a CD8 + T cell response, and there is a crucial need to refine the predictions to readily identify the best antigens that could mediate T-cell responses. Such a response requires a high enough number of epitopes bound to the target MHC. This number is correlated with both the peptide-MHC binding affinity and the number of peptides reaching the ER. Beyond this, the response may be affected by the properties of the neo-epitope mutated residues. METHODS: Herein, we analyzed several experimental datasets from cancer patients to elaborate better predictive algorithms for T-cell reactivity to neo-epitopes. RESULTS: Indeed, potent classifiers for epitopes derived from neo-antigens in melanoma and other tumors can be developed based on biochemical properties of the mutated residue, the antigen expression level and the peptide processing stage. Among MHC binding peptides, the present classifiers can remove half of the peptides falsely predicted to activate T cells while maintaining the absolute majority of reactive peptides. CONCLUSIONS: The classifier properties further highlight the contribution of the quantity of peptides reaching the ER and the mutation type to CD8 + T cell responses. These classifiers were then validated on neo-antigens obtained from other datasets, confirming the validity of our prediction. Algorithm Availability: http://peptibase.cs.biu.ac.il/Tcell_predictor/ or by request from the authors as a standalone code.

S-phase transcriptional buffering quantified on two different promoters.

Imaging of transcription by quantitative fluorescence-based techniques allows the examination of gene expression kinetics in single cells. Using a cell system for the in vivo visualization of mammalian mRNA transcriptional kinetics at single-gene resolution during the cell cycle, we previously demonstrated a reduction in transcription levels after replication. This phenomenon has been described as a homeostasis mechanism that buffers mRNA transcription levels with respect to the cell cycle stage and the number of transcribing alleles. Here, we examined how transcriptional buffering enforced during S phase affects two different promoters, the cytomegalovirus promoter versus the cyclin D1 promoter, that drive the same gene body. We found that global modulation of histone modifications could completely revert the transcription down-regulation imposed during replication. Furthermore, measuring these levels of transcriptional activity in fixed and living cells showed that the transcriptional potential of the genes was significantly higher than actual transcription levels, suggesting that promoters might normally be limited from reaching their full transcriptional potential.

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation.

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT.

Quantifying ß-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells.

Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/ß-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of ß-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear ß-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. ß-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating ß-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway.

Single-site transcription rates through fitting of ensemble-averaged data from fluorescence recovery after photobleaching: a fat-tailed distribution.

The stochastic process of gene expression is commonly controlled at the level of RNA transcription. The synthesis of messenger RNA (mRNA) is a multistep process, performed by RNA polymerase II and controlled by many transcription factors. Although mRNA transcription is intensively studied, real-time in vivo dynamic rates of a single transcribing polymerase are still not available. A popular method for examining transcription kinetics is the fluorescence recovery after photobleaching (FRAP) approach followed by kinetic modeling. Such analysis has yielded a surprisingly broad range of transcription rates. As transcription depends on many variables such as the chromatin state, binding and unbinding of transcription factors, and cell phase, transcription rates are stochastic variables. Thus, the distribution of rates is expected to follow Poissonian statistics, which does not coincide with the wide range of transcription rate results. Here we present an approach for analyzing FRAP data for single-gene transcription. We find that the transcription dynamics of a single gene can be described with a constant rate for all transcribing polymerases, while cell population transcription rates follow a fat-tailed distribution. This distribution suggests a larger probability for extreme rates than would be implied by normal distribution. Our analysis supports experimental results of transcription from two different promoters, and it explains the puzzling observation of extreme average rate values of transcription.

Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

Zooming in on single active genes in living mammalian cells.

The kinetic aspects of RNA polymerase II as it transcribes mRNA have been revealed over the past decade by use of live-cell imaging and kinetic analyses. It is now possible to visualize polymerase molecules in action, and most importantly to detect and follow the mRNA product as it is generated in real time on active genes. Questions such as the speed at which mRNAs are transcribed or the number of polymerases running along a particular gene can be addressed at high temporal resolution. These kinetic studies highlight the tight regulation that genes encounter when moving between active and inactive states, and ultimately will shed light on the kinetic aspects of transcription of genes under perturbed states. The scientific pathway along which these findings were unearthed begins with the imaging of the action of hundreds of genes working in concert in fixed cells. The state of the art has reached the capability of analyzing the transcription of single alleles in living mammalian cells.

Quantifying the transcriptional output of single alleles in single living mammalian cells.

Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization in which genes appear as single alleles. Here we present a robust technique for the quantification of mRNA synthesis from a single allele in real time in single living mammalian cells. The protocol describes how to generate cell clones harboring an MS2-tagged allele and how to detect in vivo transcription from this tagged allele at high spatial and temporal resolution throughout the cell cycle. Quantification of nascent mRNAs produced from the single tagged allele is performed using RNA fluorescence in situ hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling detailed in the protocol include measurements of transcription rates of RNA polymerase II, determination of the number of polymerases recruited to the tagged allele and measurement of the spacing between polymerases. Generation of the cells containing the single tagged alleles should take up to 1 month; RNA FISH or live-cell imaging will require an additional week.

Imaging mRNAs in living mammalian cells.

The gene expression pathway begins in the nucleus as a gene receives a cue to transcribe, and typically ends in the cytoplasm with the production of the required protein. The nuclear processes of mRNA transcription and nucleo-cytoplasmic transport are of high importance as they encompass the major control points of gene expression. While it has been possible to study the mRNA life cycle using biochemical and molecular biology approaches, the advent of methods for nucleic acid tagging in vivo, have opened up many possibilities for examining these processes in vivo. In this chapter we describe the methodology required for setting up a live-cell system for monitoring real-time mRNA dynamics in mammalian cells.

Single-allele analysis of transcription kinetics in living mammalian cells.

We generated a system for in vivo visualization and analysis of mammalian mRNA transcriptional kinetics of single alleles in real time, using single-gene integrations. We obtained high-resolution transcription measurements of a single cyclin D1 allele under endogenous or viral promoter control, including quantification of temporal kinetics of transcriptional bursting, promoter firing, nascent mRNA numbers and transcription rates during the cell cycle, and in relation to DNA replication.

Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells.

The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mRNAs. Knowledge of in vivo messenger RNA export kinetics remains poor in comparison with that of protein transport. We have established a mammalian system that allowed the real-time visualization and quantification of large single mRNA-protein complexes (mRNPs) during export. The in vivo dynamics of bulk mRNP transport and export, from transcription to the nuclear pore complex (NPC), occurred within a 5-40 minute time frame, with no NPC pile-up. mRNP export was rapid (about 0.5 s) and kinetically faster than nucleoplasmic diffusion. Export inhibition demonstrated that mRNA-NPC interactions were independent of ongoing export. Nucleoplasmic transport dynamics of intron-containing and intronless mRNAs were similar, yet an intron did increase export efficiency. Here we provide visualization and analysis at the single mRNP level of the various steps in nuclear gene expression and the inter-chromatin tracks through which mRNPs diffuse, and demonstrate the kinetics of mRNP-NPC interactions and translocation.