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Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell–derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL. Significance: Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

The list of antibodies used in the study

TIL-tumor cell coculture activated tumor-reactive T cells and generated tumor-reactive TIL-iPSCs from TIL of patient 3784. A, FACS panels showing the gating strategy to sort PD1+ 4-1BB+ CD8+ T cells after TIL-tumor cell coculture. Top panel shows the phenotype of TIL without coculture. Dump gate is a mixture of different T lineage markers (CD25, CD56, and TCRγδ) to exclude regulatory T cells (CD25+), natural killer cells (CD56+), and gamma-delta T cells (TCRγδ+). B, A summary table of the TCRβ sequence of eight preidentified tumor-reactive TCRs indicating their CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture) and in sorted PD1+ 4-1BB+ (DP) population, enrichment (DP/bulk) and presence in established iPSC clones. C, A bar graph showing the relative frequency of TCR clones, which were present in bulk population (starting cells) and reprogrammed to iPSCs, in four sorted populations [PD1− 4-1BB− (DN), PD1+ 4-1BB− (PD1+ SP), PD1− 4-1BB+ (4-1BB+ SP), PD1+ 4-1BB+ (DP)] after TIL-tumor cell coculture relative to bulk frequency of each TCR clone before coculture. Please see Supplementary Table S1 for the details of the TCR clones (TCR 2, 3, 6, 9, 11, 12, 15, and 17).

TCR stimulation is necessary for T cells to be reprogrammed to T-iPSCs. A, Schematic representation of PBMC derived T-cell reprogramming into iPSC. Whole PB T cells or the sorted populations were stimulated by αCD3/28 beads, and transduced with Sendai virus containing four Yamanaka factors and SV40 large T antigen. Approximately after 20–25 days’ culture in iPSC culture condition, the number of established iPSC colonies were counted. B, A bar graph indicates the numbers of AP-positive ES cell-like iPSC colonies derived from 1E+5 T cells with or without TCR stimulation. N = 3 for each donor. SeV containing four Yamanaka factors (OSKM) or OSKM and SV40 were used. C, Gating strategy to sort four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA). PBMC were stained by antibodies against CD3, CD8, CD62L, CD45RO, CCR7, and CD45RA. CD8+ T cells were first gated on the basis of CD45RO and CD62L expression, followed by the second gate based on CD45RA and CCR7 expression. Four subsets of CD8+ T cells (Naïve, CM, EM, and EMRA) were sorted according to the gates indicated in the flow cytometry panels. D, A summary table showing which subset (Naïve, CM, EM, and EMRA) were reprogrammed to iPSCs by different stimulation methods. Sorted CD8+ T-cell subsets (Naïve, CM, EM, and EMRA) were stimulated by different methods (OKT-3 or αCD3/28 beads) before SeV infection. E, Representative AP, phase contrast, and bright field images were shown to describe the methods of iPSC colonies enumeration from different subsets (Naïve, CM, EM, and EMRA) of CD8 T cells reprogrammed into iPSC in each experiments following transduction of Sendai virus containing four Yamanaka factors and SV40 large T antigen. Each well was seeded with 1E+5 T cells. F, A bar graph indicates the numbers of AP-positive ES cell-like colonies derived from 1E+5 sorted CD8+ naïve/CM/EM/EMRA T cells activated by αCD3/28 beads. N = 3 for each donor.

Table S1 indicating TIL-iPSCs were established from T cells of low frequency along with major tumor neoantigen specific T cells from patient 3784

TIL-tumor cell coculture resulted in establishment of TIL-iPSCs from tumor-reactive T-cell clones. A, Schematic representation of TIL-tumor cell coculture to isolate tumor-reactive T cells and subsequent reprogramming into iPSCs. Autologous tumor cell line and minimally cultured TIL were cocultured for 16 hours, sorted for PD1+ 4-1BB+ CD8+ T cells, and transduced with Sendai virus containing four Yamanaka factors and SV40 large T antigen. On days 20–21 when cells formed domed shaped ES cell-like colonies, they were individually collected as clones and expanded. B, A summary table of nine different TCRs identified from 13 different master tubes containing 221 TIL-iPSC clones by using immunosequencing analysis. CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture in starting material) and in sorted PD1+ 4-1BB+ (DP) population, enrichment (DP/Bulk), the least number of iPSC clones established and the type of TCR were noted. PIR, preidentified reactive TCR; NF, newly found TCR (unknown reactivity). C, A summary table of the TCRβ sequence analysis of six preidentified tumor-reactive TCRs (PIR-TCR I–VI) indicating the CDR3β amino acid sequence, Vβ family, frequency in bulk (before coculture) and in the sorted PD1+ 4-1BB+ (DP) population after TIL-tumor cell coculture, enrichment (DP/Bulk) and presence in established iPSC clones. * means the TCR was not found in established T-iPSC clones but detected in the remaining clones in the dish after picking up iPS cell colonies (mother dish). D, A graph indicates the frequency of PIR-TCRs in starting cells (Bulk) and in sorted PD1+ 4-1BB+ (DP) cells. E, A bar graph indicates the enrichment analysis (the ratio of DP/Bulk) of those six PIR-TCRs.

Nonspecific TCR stimulation by αCD3 Ab is not suitable for selective reprogramming of tumor-reactive T cells. A, A summary table of top 10 frequent TCRβ sequences in the starting cells, that is, expanded TIL of patient 4069, indicating the CDR3β amino acid sequence, Vβ family and frequency. The most frequent TCR was the patient's neoantigen-specific TCR (indicated in red). B, A summary table of TCRβ sequences of established TIL-iPSCs by αCD3 Ab stimulation, indicating the CDR3β amino acid sequence, Vβ family and the number of TIL-iPSC clones established. C, A summary table of six candidate TCR pairs including five newly found TCRs (NF TCR 1–5) and one PIR-TCR which were sequenced individually from master tube 6 containing 14 individual iPSC clones. The table indicates TCR pairs, CDR3β sequence, Vβ family, CDR3α sequence, and Vα family. NF, newly found TCR (unknown reactivity); PIR, preidentified reactive TCR. D and E, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous DC pulsed with wild-type (WT) peptide (16 mer or 9 mer) or mutant peptide (9 mer). D shows the representative FACS plots and E shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. F and G, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous, or HLA-matched allogeneic tumor cells derived from the PDXs model. F shows the representative FACS plots and G shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. H, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed PDX-derived tumor cell line cultured with T cells transduced with PIR-TCR analyzed by the Incucyte live imaging and analysis system (effector-to-target ratio of 2:1). Empty vector transduced T cells (Mock) and tumor cell alone (TC) were used as negative controls. The results are shown as mean ± SD (N = 4).

Tumor-infiltrating lymphocytes (TIL) that can recognize and kill tumor cells have curative potential in subsets of patients treated with adoptive cell transfer (ACT). However, lack of TIL therapeutic efficacy in many patients may be due in large part to a paucity of tumor-reactive T cells in TIL and the exhausted and terminally differentiated status of those tumor-reactive T cells. We sought to reprogram exhausted TIL that possess T-cell receptors (TCR) specific for tumor antigens into induced pluripotent stem cells (iPSC) to rejuvenate them for more potent ACT. We first attempted to reprogram tumor neoantigen-specific TIL by αCD3 Ab prestimulation which resulted in failure of establishing tumor-reactive TIL-iPSCs, instead, T cell–derived iPSCs from bystander T cells were established. To selectively activate and enrich tumor-reactive T cells from the heterogenous TIL population, CD8+ PD-1+ 4-1BB+ TIL population were isolated after coculture with autologous tumor cells, followed by direct reprogramming into iPSCs. TCR sequencing analysis of the resulting iPSC clones revealed that reprogrammed TIL-iPSCs encoded TCRs that were identical to the pre-identified tumor-reactive TCRs found in minimally cultured TIL. Moreover, reprogrammed TIL-iPSCs contained rare tumor antigen-specific TCRs, which were not detectable by TCR sequencing of the starting cell population. Thus, reprogramming of PD-1+ 4-1BB+ TIL after coculture with autologous tumor cells selectively generates tumor antigen-specific TIL-iPSCs, and is a distinctive method to enrich and identify tumor antigen-specific TCRs of low frequency from TIL.Significance:Reprogramming of TIL into iPSC holds great promise for the future treatment of cancer due to their rejuvenated nature and the retention of tumor-specific TCRs. One limitation is the lack of selective and efficient methods for reprogramming tumor-specific T cells from polyclonal TIL. Here we addressed this limitation and present a method to efficiently reprogram TIL into iPSC colonies carrying diverse tumor antigen reactive TCR recombination.

TIL stimulated with autologous tumor cell line generated antigen specific iPSC whereas αCD3/28 beads mediated stimulation did not. A, Summary of three candidate TCR pairs identified in TIL-iPSC lines established by different stimulation methods. Bead Stim TCR: The most dominant clone in the TIL-iPSCs established by αCD3/28 beads stimulation, Tumor Stim TCR: The TCR identified in the TIL-iPSC clones established by coculture with autologous tumor cells (TCR 4 in Fig. 3B), and PIR-TCR III: preidentified reactive TCR clone detected in the TIL-iPSC clones established by coculture with autologous tumor cells. The table includes TCR type, CDR3β amino acid sequences, Vβ family, CDR3α amino acid sequences, Vα family are described. B and C, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 4A). T cells were cocultured with autologous or allogeneic tumor cell lines. B shows the representative FACS plots and C shows the percentage of 4-1BB+ cells in each condition. N = 3 for each condition. Representative data of four independent experiments. Mock: empty vector transduced PBL as a negative control, PIR-TCR III as a positive control. D, A bar graph showing the results of ELISA IFNγ production assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 4A). T cells were cocultured with autologous or allogeneic tumor cell lines for 16 hours and culture soup were analyzed for IFNγ by ELISA. N = 3 for each condition. Representative data of four independent experiments. E, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed tumor cells from pt. 1913 cocultured with different TCRs (listed in Fig. 4A), analyzed by the Incucyte live imaging and analysis system at effector-to-target ratio of 2:1. Mock indicates empty vector transduced T cells used as a negative control. The results are shown as the mean ± SD of N = 4 each condition. A representative data of four independent experiments.

Figure S1: A summary table of the patient samples used and the schema of the study

Summary: Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from naïve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8αβ(+) antigen-specific T cells with a naïve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

Despite the long-standing observation that herpes simplex virus (HSV) latency-associated transcript (LAT) promoter deletion viruses show impaired recurrence phenotypes in relevant animal models, the mechanism by which these sequences exert this phenotypic effect is unknown. We constructed and evaluated four mutant HSV-2 isolates with targeted mutations in the LAT promoter and LAT-associated microRNAs (miRNAs) affecting (i) the LAT TATA box; (ii) the LAT ICP4-binding site; (iii) miRNA I (miR-I) and miR-II (miR-I/II), which both target ICP34.5; and (iv) miR-III, which targets ICP0. While the LAT TATA box mutant caused milder acute infections than wild-type (WT) virus, there was no difference in the recurrence phenotype between these viruses. LAT and miRNA expression during latency was not impaired by this mutation, suggesting that other promoter elements may be more important for latent HSV-2 LAT expression. Mutation of the LAT ICP4-binding site also did not cause an in vivo phenotypic difference between mutant and WT viruses. Acute infection and reactivation from latency of the miR-I/II mutant were similar to those of its rescuant, although the acute infection was slightly reduced in severity relative to that caused by the wild-type virus. The miR-III mutant also exhibited WT phenotypes in acute and recurrent phases of infection. While they do not rule out an effect of these elements in human latency or reactivation, these findings do not identify a specific role for LAT or LAT-associated miRNAs in the HSV-2 LAT promoter deletion phenotype in guinea pigs. Thus, other sequences in this region may play a more important role in the long-studied LAT-associated phenotype in animals. IMPORTANCE While it has been known for several decades that specific HSV-1 and HSV-2 sequences near the LAT promoter are required for efficient viral reactivation in animal models, the mechanism is still not known. We constructed four mutant viruses with the goal of identifying critical sequence elements and of specifically testing the hypothesis that microRNAs that are expressed during latency play a role. Determination that specific LAT promoter sequences and miRNA sequences do not influence viral reactivation of HSV-2 helps to narrow down the search for the mechanism by which the virus controls its latency and recurrence phenotype.

T cells are potentially curative for patients with metastatic cancer, but many patients with cancer have T cells that are 'terminally differentiated', a condition associated with treatment failure. We have observed that less differentiated T cells have a greater capacity to proliferate, persist and destroy large cancer deposits. Advances in regenerative medicine might allow the generation of rejuvenated T cells from induced pluripotent stem cells (iPSC). We have previously reported that T cells can be generated from iPSC in vitro by co-culturing them OP9 stromal cells expressing Notch-1 ligand, Delta-like-1 (OP9/DLL1). These cells have limited tumor-specificity but also exhibit unconventional and NK cell-like properties demonstrating lineage diversion into alternative lymphoid development pathways, with unknown consequences for their safety and efficacy. To generate iPSC-derived T cells with more naturalistic tumor-specific T cell programs, we sought to restore physiologic signals for selection, maturation and survival. We employed a novel 3D thymic culture system using fetal thymic tissue and generated a novel type of T cell, 'iPSC-derived thymic emigrants' (iTE). Antigen-specific CD8αβ+ iTE exhibited functional properties in vitro that were almost indistinguishable from natural naïve CD8αβ+ T cells, including vigorous expansion and robust anti-tumor activity. iPSC-derived immature T cells generated using OP9/DLL1 and 'educated' in fetal thymic organoids in a 3D culture system resembled naturally-occurring 'young' T cells, as analyzed using whole genome RNA-seq techniques. iTE recapitulated many of the transcriptional programs of naïve T cells in vivo and revealed a striking capacity for engraftment, memory formation and efficient tumor destruction. Although many milestones remain, our data show that 'Next-Gen' autologous tumor-specific T cells can realistically be generated from iPSC using 3D thymic organ tissue. Our next goal is now to employ these cells to treat patients with metastatic cancer because iPSC-derived T cells have a potentially unlimited capacity for proliferation, engraftment and anti-tumor activity. Disclosures Vizcardo: NCI: Patents & Royalties: International Patent Application PCT/US2017/65986. Klemen:NIH/NCI: Patents & Royalties: International Patent Application PCT/US2017/65986. Restifo:NIH/NCI: Patents & Royalties: International Patent Application PCT/US2017/65986.

The high prevalence of Herpesviruses in the population and the maintenance of lifelong latent reservoirs are challenges to the control of herpetic diseases, despite the availability of antiviral pharmaceuticals that target viral DNA replication. In addition to oral and genital lesions, herpes simplex virus infections and recurrent reactivations from the latent pool can result in severe pathology including neonatal infection and mortality, blindness due to ocular keratitis, and viral-induced complications in immunosuppressed individuals. Herpesviruses, like their cellular hosts, are subject to the regulatory impacts of chromatin and chromatin modulation machinery that promotes or suppresses gene expression. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 and JMJD2s. Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and a block to infection and reactivation in vitro. Here, the concept of epigenetic suppression of viral infection is demonstrated in three animal models of herpes simplex virus infection and disease. Inhibition of LSD1 via treatment of animals with the monoamine oxidase inhibitor tranylcypromine results in suppression of viral lytic infection, subclinical shedding, and reactivation from latency in vivo. Phenotypic suppression is correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Given the expanding development of epipharmaceuticals, this approach has substantial potential for anti-herpetic treatments with distinct advantages over the present pharmaceutical options.

In order to understand factors that may influence latency-associated transcription and latency-associated transcript (LAT) phenotypes, we studied the expression of the herpes simplex virus 2 (HSV-2) LAT-associated microRNAs (miRNAs). We mapped the transcription initiation sites of all three primary miRNA transcripts and identified the ICP4-binding sequences at the transcription initiation sites of both HSV-2 LAT (pri-miRNA for miR-I and miR-II, which target ICP34.5, and miR-III, which targets ICP0) and L/ST (a pri-miRNA for miR-I and miR-II) but not at that of the primary miR-H6 (for which the target is unknown). We confirmed activity of the putative HSV-2 L/ST promoter and found that ICP4 trans -activates the L/ST promoter when the ICP4-binding site at its transcription initiation site is mutated, suggesting that ICP4 may play a dual role in regulating transcription of L/ST and, consequently, of miR-I and miR-II. LAT exon 1 (containing LAT enhancer sequences), together with the LAT promoter region, comprises a bidirectional promoter required for the expression of both LAT-encoded miRNAs and miR-H6 in latently infected mouse ganglia. The ability of ICP4 to suppress ICP34.5-targeting miRNAs and to activate lytic viral genes suggests that ICP4 could play a key role in the switch between latency and reactivation. IMPORTANCE The HSV-2 LAT and viral miRNAs expressed in the LAT region are the most abundant viral transcripts during HSV latency. The balance between the expression of LAT and LAT-associated miRNAs and the expression of lytic viral transcripts from the opposite strand appears to influence whether individual HSV-infected neurons will be latently or productively infected. The outcome of neuronal infection may thus depend on regulation of gene expression of the corresponding primary miRNAs. In the present study, we characterize promoter sequences responsible for miRNA expression, including identification of the primary miRNA 5′ ends and evaluation of ICP4 response. These findings provide further insight into the virus' strategy to tightly control expression of lytic cycle genes (especially the neurovirulence factor, ICP34.5) and suggest a mechanism (via ICP4) for the transition from latency to reactivated productive infection.