FOXO1 Inhibition Generates Potent Nonactivated CAR T Cells against Solid Tumors.

Chimeric antigen receptor (CAR) T cells have shown promising results in the treatment of B-cell malignancies. Despite the successes, challenges remain. One of them directly involves the CAR T-cell manufacturing process and especially the ex vivo activation phase. While this is required to allow infection and expansion, ex vivo activation dampens the antitumor potential of CAR T cells. Optimizing the nature of the T cells harboring the CAR is a strategy to address this obstacle and has the potential to improve CAR T-cell therapy, including for solid tumors. Here, we describe a protocol to create CAR T cells without ex vivo preactivation by inhibiting the transcription factor FOXO1 (CAR TAS cells). This approach made T cells directly permissive to lentiviral infection, allowing CAR expression, with enhanced antitumor functions. FOXO1 inhibition in primary T cells (TAS cells) correlated with acquisition of a stem cell memory phenotype, high levels of granzyme B, and increased production of TNFα. TAS cells displayed enhanced proliferative and cytotoxic capacities as well as improved migratory properties. In vivo experiments showed that CAR TAS cells were more efficient at controlling solid tumor growth than classical CAR T cells. The production of CAR TAS from patients' cells confirmed the feasibility of the protocol in clinic.

FOXO1 transcription factor plays a key role in T cell-HIV-1 interaction.

HIV-1 is dependent on the host cell for providing the metabolic resources for completion of its viral replication cycle. Thus, HIV-1 replicates efficiently only in activated CD4+ T cells. Barriers preventing HIV-1 replication in resting CD4+ T cells include a block that limits reverse transcription and also the lack of activity of several inducible transcription factors, such as NF-κB and NFAT. Because FOXO1 is a master regulator of T cell functions, we studied the effect of its inhibition on T cell/HIV-1 interactions. By using AS1842856, a FOXO1 pharmacologic inhibitor, we observe that FOXO1 inhibition induces a metabolic activation of T cells with a G0/G1 transition in the absence of any stimulatory signal. One parallel outcome of this change is the inhibition of the activity of the HIV restriction factor SAMHD1 and the activation of the NFAT pathway. FOXO1 inhibition by AS1842856 makes resting T cells permissive to HIV-1 infection. In addition, we found that FOXO1 inhibition by either AS1842856 treatment or upon FOXO1 knockdown induces the reactivation of HIV-1 latent proviruses in T cells. We conclude that FOXO1 has a central role in the HIV-1/T cell interaction and that inhibiting FOXO1 with drugs such as AS1842856 may be a new therapeutic shock-and-kill strategy to eliminate the HIV-1 reservoir in human T cells.

Fam65b Phosphorylation Relieves Tonic RhoA Inhibition During T Cell Migration.

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse, we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding, at the molecular level, how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here, we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally, we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization, as well as T cell migration in vitro. Altogether, our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

FAM65B controls the proliferation of transformed and primary T cells.

Cell quiescence is controlled by regulated genome-encoded programs that actively express genes which are often down-regulated or inactivated in transformed cells. Among them is FoxO1, a transcription factor that imposes quiescence in several cell types, including T lymphocytes. In these cells, the FAM65B encoding gene is a major target of FOXO1. Here, we show that forced expression of FAM65B in transformed cells blocks their mitosis because of a defect of the mitotic spindle, leading to G2 cell cycle arrest and apoptosis. Upon cell proliferation arrest, FAM65B is engaged in a complex containing two proteins well known to be involved in cell proliferation i.e. the HDAC6 deacetylase and the 14.3.3 scaffolding protein. In primary T cells, FAM65B is down-regulated upon T cell receptor engagement, and maintaining its expression blocks their proliferation, establishing that the decrease of FAM65B expression is required for proliferation. Conversely, inhibiting FAM65B expression in naive T lymphocytes decreases their activation threshold. These results identify FAM65B as a potential new target for controlling proliferation of both transformed and normal cells.

Evidence that HIV-1 restriction factor SAMHD1 facilitates differentiation of myeloid THP-1 cells.

BACKGROUND: SAMHD1 counteracts HIV-1 or HIV-2/SIVsmm that lacks Vpx by depleting the intracellular pool of nucleotides in myeloid cells and CD4+ quiescent T cells, thereby inhibiting the synthesis of retroviral DNA by reverse transcriptase. Depletion of nucleotides has been shown to underline the establishment of quiescence in certain cellular systems. These observations led us to investigate whether SAMHD1 could control the transition between proliferation and quiescence using the THP-1 cell model. FINDINGS: The entry of dividing THP-1 myeloid cells into a non-dividing differentiated state was monitored after addition of phorbol-12-myristate-13-acetate (PMA), an inducer of differentiation. Under PMA treatment, cells overexpressing SAMHD1 display stronger and faster adhesion to their support, compared to cells expressing a catalytically inactive form of SAMHD1, or cells depleted of SAMHD1, which appear less differentiated. After PMA removal, cells overexpressing SAMHD1 maintain low levels of cyclin A, in contrast to other cell lines. Interestingly, SAMHD1 overexpression slightly increases cell adhesion even in the absence of the differentiation inducer PMA. Finally, we found that levels of SAMHD1 are reduced in proliferating primary CD4+ T cells after T cell receptor activation, suggesting that SAMHD1 may also be involved in the transition from a quiescent state to a dividing state in primary T cells. CONCLUSIONS: Altogether, we provide evidence that SAMHD1 may facilitate some aspects of THP-1 cell differentiation. Restriction of HIV-1 by SAMHD1 may rely upon its ability to modify cell cycle parameters, in addition to the direct inhibition of reverse transcription.

A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.

Forkhead box O (FOXO) transcription factors favor both T cell quiescence and trafficking through their control of the expression of genes involved in cell cycle progression, adhesion, and homing. In this article, we report that the product of the fam65b gene is a new transcriptional target of FOXO1 that regulates RhoA activity. We show that family with sequence similarity 65 member b (Fam65b) binds the small GTPase RhoA via a noncanonical domain and represses its activity by decreasing its GTP loading. As a consequence, Fam65b negatively regulates chemokine-induced responses, such as adhesion, morphological polarization, and migration. These results show the existence of a new functional link between FOXO1 and RhoA pathways, through which the FOXO1 target Fam65b tonically dampens chemokine-induced migration by repressing RhoA activity.

Retroviral infection in vivo requires an immune escape virulence factor encrypted in the envelope protein of oncoretroviruses.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins (Envs). The envelope-mediated immunosuppression was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation specifically abolishes IS activity without affecting the "mechanical" fusogenic function of the entire envelope. Here, we genetically "switched off' the envelope-mediated immunosuppression of an infectious retrovirus, the Friend murine leukemia virus, while preserving mutant envelope infectivity both ex vivo and in vivo, thus allowing us to test the functional importance of envelope-mediated immunosuppression in retrovirus physiology. Remarkably, we show, in vivo, that the non-IS mutant virus displays the same propagation kinetics as its WT counterpart in irradiated immunocompromised mice but that it is rapidly and totally cleared from normal immunocompetent mice, which become fully protected against a challenge with the WT retrovirus. Using cell depletion strategies, we further establish that envelope-mediated immunosuppression enables the retrovirus to escape innate (natural killer cells) and adaptive (CD8 T cells) antiviral effectors. Finally, we show that inactivated mutant virions induce higher humoral and cellular responses than their WT counterparts. In conclusion, our work demonstrates the critical role of Env-induced immunosuppression for retrovirus propagation in vivo and identifies a unique definite target for antiretroviral therapies and vaccine strategies, also characterized in the human T-cell leukemia virus (HTLV) and xenotropic murine leukemia virus-related virus (XMRV) retroviruses, opening unprecedented prospects for the treatment of retroviral diseases.

FOXO1 regulates L-Selectin and a network of human T cell homing molecules downstream of phosphatidylinositol 3-kinase.

In T cells, the PI3K pathway promotes proliferation and survival induced by Ag or growth factors, in part by inactivating the FOXO transcription factor 1. We now report that FOXO1 controls the expression of L-selectin, an essential homing molecule, in human T lymphocytes. This control is already operational in unprimed T cells and involves a transcriptional regulation process that requires the FOXO1 DNA-binding domain. Using transcriptional profiling, we demonstrate that FOXO1 also increases transcripts of EDG1 and EDG6, two sphingosine-1-phosphate receptors that regulate lymphocyte trafficking. Additionally, FOXO1 binds the promoter of the cell quiescence and homing regulator Krüppel-like factor 2 and regulates its expression. Together, these results reveal a new function of FOXO1 in the immune system and suggest that PI3K controls a coordinated network of transcription factors regulating both cell quiescence and homing of human T lymphocytes.

Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins.

We have previously demonstrated that the envelope proteins of a murine and primate retrovirus are immunosuppressive in vivo. This property was manifested by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to have the env-expressing cells escape (at least transiently) immune rejection. Here, we analyzed the immunosuppressive activity of the human and murine syncytins. These are envelope genes from endogenous retroviruses independently coopted by ancestral hosts, conserved in evolution, specifically expressed in the placenta, and with a cell-cell fusogenic activity likely contributing to placenta morphogenesis. We show that in both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is immunosuppressive and, rather unexpectedly, the other (human syncytin-1 and mouse syncytin-A) is not (albeit able to induce cell-cell fusion). Delineation of the immunosuppressive domain by deletion analysis, combined with a comparison between immunosuppressive and nonimmunosuppressive sequences, allowed us to derive a mutation rule targeted to specific amino acids, resulting in selective switch from immunosuppressive to nonimmunosuppressive envelope proteins and vice versa. These results unravel a critical function of retroviral envelopes, not necessarily "individually" selected for in the retrovirus endogenization process, albeit "tandemly" conserved in evolution for the syncytin pairs in primates and Muridae. Selective inactivation of immunosuppression, under conditions not affecting fusogenicity, should be important for understanding the role of this function in placental physiology and maternofetal tolerance.

Mobility and integration sites of a murine C57BL/6 melanoma endogenous retrovirus involved in tumor progression in vivo.

Tumor development is a multistep process in which both genetic and epigenetic events cooperate for the emergence of a malignant clone with metastatic properties. The possibility that endogenous retroviruses promote the expansion of a neoplastic clone by subverting immunosurveillance has been proposed and recently demonstrated in the case of the B16 murine melanoma, which spontaneously express the melanoma-associated retrovirus (MelARV). Indeed, knocking down, by RNA interference, this endogenous retrovirus resulted in the rejection of the tumor cells in immunocompetent mice, without any alteration of their transformed phenotype. Here, we characterize the MelARV proviruses present in the B16 melanoma. Complete sequencing of the viral genomic RNA and characterization of the integration sites within both the B16 tumor cells and a subline selected in vivo for increased metastatic activity disclosed mobility of the element with new proviral insertions targeting critical genes and altering their transcriptional profile. The results show that MelARV can act both at the genetic level, inducing mutations by insertion, and at the epigenetic level, promoting immunosuppression of the host. These properties may as well be relevant to human tumors, such as germline tumors and melanoma, where endogenous retroviruses are active.

A recombinant endogenous retrovirus amplified in a mouse neuroblastoma is involved in tumor growth in vivo.

The theory of immunoediting postulates that tumor cells exhibit a reduced immunogenicity to escape eradication by the host immune system. It has been proposed that endogenous retroviruses--provided that they are active--could play a role in this process, via the immunosuppressive domain carried by their envelope protein. Here, we demonstrate that the Neuro-2a tumor cell line--originating from a spontaneous A/J mouse neuroblastoma--produces an infectious retrovirus that most probably results from a recombination event between 2 mouse endogenous retroviral elements. This Neuro-2a-associated recombinant retrovirus derives from the unique ecotropic provirus located at the Emv-1 locus, but with a gag sequence conferring B-tropism, thus allowing its high-level amplification in Neuro-2a cells. We show that knocking down -by RNA interference- this endogenous retrovirus in Neuro-2a cells has no effect on the transformed phenotype of the cells, but results in delayed tumor growth and prolonged animal survival, following engraftment of the cells into immunocompetent mice. Recombination between endogenous retroviruses, amplification of the resulting element and high-level expression of its immunosuppressive activity are therefore likely steps of an immunoediting process, leading to an invading tumor.

Endogenous retrovirus expression is required for murine melanoma tumor growth in vivo.

Tumor development is a multistep process in which both genetic and epigenetic events cooperate for the emergence of a malignant clone. The possibility that endogenous retroviruses promote the expansion of a neoplastic clone by subverting immune surveillance has been proposed, but remained elusive. Here we show that knocking down-by RNA interference-an endogenous retrovirus spontaneously induced in the B16 murine melanoma results in the rejection of the tumor cells in immunocompetent mice, under conditions where control melanoma cells grow into lethal tumors. The knockdown does not modify the transformed phenotype of the cells, as measured both in vitro by a soft agar assay and in vivo by tumor cell proliferation in immunoincompetent (X-irradiated and severe combined immunodeficiency) mice. Tumor rejection can be reverted upon adoptive transfer of regulatory T cells from control melanoma-engrafted mice, as well as upon reexpression of the sole envelope gene of the endogenous retrovirus in the knocked down cells. These results show that endogenous retroviruses can be essential for a regulatory T-cell-mediated subversion of immune surveillance and could be relevant to human tumors where such elements-and especially their envelope gene-are induced.

The full-length envelope of an HERV-H human endogenous retrovirus has immunosuppressive properties.

We have demonstrated previously that the envelope proteins of a murine retrovirus (Moloney murine leukaemia virus) and a simian retrovirus (Mason-Pfizer monkey virus) have immunosuppressive properties in vivo. This property was manifested by the ability of the proteins, when expressed by tumour cells normally rejected by engrafted mice, to allow the envelope-expressing cells to escape immune rejection and to proliferate. Here, it is shown that this property is not restricted to the envelope of infectious retroviruses, but is also shared by the envelope protein encoded by an endogenous retrovirus of humans belonging to the HERV-H family. These results emphasize the close relationship between endogenous and infectious retroviruses and might be important in relation to the process of tumour progression in humans.

The envelope of Mason-Pfizer monkey virus has immunosuppressive properties.

We have demonstrated previously that the envelope protein of a murine retrovirus, Moloney murine leukaemia virus, has immunosuppressive properties in vivo. This property was manifested by the ability of the protein, when expressed by tumour cells normally rejected by engrafted mice, to allow the env-expressing cells to escape immune rejection and to proliferate. Here, it is shown that this property is not restricted to the envelope of a murine retrovirus, but is also shared by the envelope encoded by a primate retrovirus, Mason-Pfizer monkey virus.