Latent human herpesvirus 6 is reactivated in CAR T cells.

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells.

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.

Preclinical Evaluation of Allogeneic CAR T Cells Targeting BCMA for the Treatment of Multiple Myeloma.

Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.

A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies.

CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety.

Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy.

Both in vivo data in preclinical cancer models and in vitro data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties in vivo.

Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.

The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease.

ICOS Promotes the Function of CD4+ Effector T Cells during Anti-OX40-Mediated Tumor Rejection.

ICOS is a T-cell coregulatory receptor that provides a costimulatory signal to T cells during antigen-mediated activation. Antitumor immunity can be improved by ICOS-targeting therapies, but their mechanism of action remains unclear. Here, we define the role of ICOS signaling in antitumor immunity using a blocking, nondepleting antibody against ICOS ligand (ICOS-L). ICOS signaling provided critical support for the effector function of CD4(+) Foxp3(-) T cells during anti-OX40-driven tumor immune responses. By itself, ICOS-L blockade reduced accumulation of intratumoral T regulatory cells (Treg), but it was insufficient to substantially inhibit tumor growth. Furthermore, it did not impede antitumor responses mediated by anti-4-1BB-driven CD8(+) T cells. We found that anti-OX40 efficacy, which is based on Treg depletion and to a large degree on CD4(+) effector T cell (Teff) responses, was impaired with ICOS-L blockade. In contrast, the provision of additional ICOS signaling through direct ICOS-L expression by tumor cells enhanced tumor rejection and survival when administered along with anti-OX40 therapy. Taken together, our results showed that ICOS signaling during antitumor responses acts on both Teff and Treg cells, which have opposing roles in promoting immune activation. Thus, effective therapies targeting the ICOS pathway should seek to promote ICOS signaling specifically in effector CD4(+) T cells by combining ICOS agonism and Treg depletion. Cancer Res; 76(13); 3684-9. ©2016 AACR.

Identification of a novel mechanism of action of fingolimod (FTY720) on human effector T cell function through TCF-1 upregulation.

BACKGROUND: Fingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function. METHODS: T cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3ß. RESULTS: We showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients. CONCLUSIONS: These results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function.

Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa.

BACKGROUND: Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. METHODS AND FINDINGS: Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I-presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. CONCLUSIONS: These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades NK-cell-mediated immune pressure and the functional validation of a structural modeling approach will facilitate the development of novel targeted immune interventions to harness the antiviral activities of NK cells.

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration.

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.

A pharmacogenetic study implicates SLC9a9 in multiple sclerosis disease activity.

OBJECTIVE: A proportion of multiple sclerosis (MS) patients experience disease activity despite treatment. The early identification of the most effective drug is critical to impact long-term outcome and to move toward a personalized approach. The aim of the present study is to identify biomarkers for further clinical development and to yield insights into the pathophysiology of disease activity. METHODS: We performed a genome-wide association study in interferon-ß (IFNß)-treated MS patients followed by validation in 3 independent cohorts. The role of the validated variant was examined in several RNA data sets, and the function of the presumed target gene was explored using an RNA interference approach in primary T cells in vitro. RESULTS: We found an association between rs9828519(G) and nonresponse to IFNß (pdiscovery = 4.43 × 10(-8)) and confirmed it in a meta-analysis across 3 replication data sets (preplication = 7.78 × 10(-4)). Only 1 gene is found in the linkage disequilibrium block containing rs9828519: SLC9A9. Exploring the function of this gene, we see that SLC9A9 mRNA expression is diminished in MS subjects who are more likely to have relapses. Moreover, SLC9A9 knockdown in T cells in vitro leads an increase in expression of IFNγ, which is a proinflammatory molecule. INTERPRETATION: This study identifies and validates the role of rs9828519, an intronic variant in SLC9A9, in IFNß-treated subjects, demonstrating a successful pharmacogenetic screen in MS. Functional characterization suggests that SLC9A9, an Na(+) -H(+) exchanger found in endosomes, appears to influence the differentiation of T cells to a proinflammatory fate and may have a broader role in MS disease activity, outside of IFNß treatment.

An IL-27/NFIL3 signalling axis drives Tim-3 and IL-10 expression and T-cell dysfunction.

The inhibitory receptor T-cell immunoglobulin and mucin domain-3 (Tim-3) has emerged as a critical regulator of the T-cell dysfunction that develops in chronic viral infections and cancers. However, little is known regarding the signalling pathways that drive Tim-3 expression. Here, we demonstrate that interleukin (IL)-27 induces nuclear factor, interleukin 3 regulated (NFIL3), which promotes permissive chromatin remodelling of the Tim-3 locus and induces Tim-3 expression together with the immunosuppressive cytokine IL-10. We further show that the IL-27/NFIL3 signalling axis is crucial for the induction of Tim-3 in vivo. IL-27-conditioned T helper 1 cells exhibit reduced effector function and are poor mediators of intestinal inflammation. This inhibitory effect is NFIL3 dependent. In contrast, tumour-infiltrating lymphocytes from IL-27R(-/-) mice exhibit reduced NFIL3, less Tim-3 expression and failure to develop dysfunctional phenotype, resulting in better tumour growth control. Thus, our data identify an IL-27/NFIL3 signalling axis as a key regulator of effector T-cell responses via induction of Tim-3, IL-10 and T-cell dysfunction.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Dysfunctional HIV-specific CD8+ T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis.

Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.

Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents.

RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene's phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions.

The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication.

The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction.