A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Expression of inducible factors reprograms CAR-T cells for enhanced function and safety.

Despite the success of CAR-T cell cancer immunotherapy, challenges in efficacy and safety remain. Investigators have begun to enhance CAR-T cells with the expression of accessory molecules to address these challenges. Current systems rely on constitutive transgene expression or multiple viral vectors, resulting in unregulated response and product heterogeneity. Here, we develop a genetic platform that combines autonomous antigen-induced production of an accessory molecule with constitutive CAR expression in a single lentiviral vector called Uni-Vect. The broad therapeutic application of Uni-Vect is demonstrated in vivo by activation-dependent expression of (1) an immunostimulatory cytokine that improves efficacy, (2) an antibody that ameliorates cytokine-release syndrome, and (3) transcription factors that modulate T cell biology. Uni-Vect is also implemented as a platform to characterize immune receptors. Overall, we demonstrate that Uni-Vect provides a foundation for a more clinically actionable next-generation cellular immunotherapy.

Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting.

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.

Preclinical evaluation of cancer immune therapy using patient-derived tumor antigen-specific T cells in a novel xenograft platform.

OBJECTIVES: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel in vivo model (called Xenomimetic or 'X' mouse) that allows monitoring of the ability of human tumor-specific T cells to suppress tumor growth following their entry into the tumor. METHODS: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD-scid IL2Rγnull (NSG) mice following an intraperitoneal injection of melanoma target cells expressing tumor neoantigen peptides, as well as green fluorescent protein and/or luciferase. Changes in tumor burden, as well as in the number and phenotype of adoptively transferred patient-derived tumor neoantigen-specific T cells in response to immunotherapy, are measured by imaging to detect fluorescence/luminescence and flow cytometry, respectively. RESULTS: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells. CONCLUSION: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies.

Abnormal immunological profile and vaginal microbiota in women prone to urinary tract infections.

The host determinants of susceptibility to recurrent urinary tract infections (UTI) are poorly understood. We investigated whether the susceptibility is associated with abnormalities in the immunological defense and further explored the linkage to vaginal microbiota. For this purpose, we compared vaginal, urine, and blood samples collected during a disease-free period from 22 women with recurrent UTI and from 17 controls. In UTI-prone women, interleukin-12 (IL-12) production in peripheral monocytes and myeloid dendritic cells (DCs) was significantly (P < 0.05) enhanced whether measured in relative numbers of IL-12-producing cells or in mean IL-12 production per cell. In contrast, no T-cell polarization was observed. Interestingly, it seemed that the cytokine production of DCs and monocytes did not translate into T-cell activation in the UTI-prone group in a manner similar to that seen with the controls. In vaginal mucosa, UTI-prone women had a lower concentration of tissue repair-associated vascular endothelial growth factor (VEGF) (P = 0.006) and less often had detectable amounts of the chief monocyte and DC chemoattractant, monocyte chemotactic protein 1 (P = 0.005), than the controls. The microbiota of UTI-prone women was characterized by a diminished lactobacillus morphotype composition, with an abnormally high (>3) mean Nugent score of 4.6 compared to 1.7 for the controls (P = 0.003). Normal lactobacillus composition was associated with increased IL-17 and VEGF concentrations in vaginal mucosa. In conclusion, immunological defects and a persistently aberrant microbiota, a lack of lactobacilli in particular, may contribute to susceptibility to recurrent UTI. Further studies of antigen-presenting-cell function and T-cell activation in recurrent UTI are called for.

Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

Human embryonic stem cells possess immune-privileged properties.

Human embryonic stem cells (hESCs) are envisioned to be a major source for cell-based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Surprisingly, immune responses to hESCs have yet to be experimentally evaluated. Here, injection of hESCs into immune-competent mice was unable to induce an immune response. Undifferentiated and differentiated hESCs failed to stimulate proliferation of alloreactive primary human T cells and inhibited third-party allogeneic dendritic cell-mediated T-cell proliferation via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs were still responsive to allogeneic stimulators but failed to proliferate upon re-exposure to hESCs. Our study demonstrates that hESCs possess unique immune-privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESCs that may avoid immune-mediated rejection.

Viewpoint: therapeutic implications of CTLA-4 compartmentalization.

Understanding the regulatory events involved in the activation and inactivation of T cells is crucial to develop therapeutic approaches for autoimmune diseases and for organ transplantation. Co-stimulatory signals delivered through the CD28 receptor and inhibitory signals through CTLA-4 are required for the proper modulation of T cell responses and the induction and maintenance of peripheral tolerance. Manipulation of these signals is emerging as a potential strategy to prevent allograft rejection in different animal models. Recent data on the compartmentalization and the structural features of CTLA-4 within T cells provides critical information not only on the molecular basis of T cell inactivation by CTLA-4, but also on the key requirements for the successful development of therapeutic strategies targeting this molecule.

Inhibition of CTLA-4 function by the regulatory subunit of serine/threonine phosphatase 2A.

The catalytic subunit of the serine/threonine phosphatase 2A (PP2A) can interact with the cytoplasmic tail of CTLA-4. However, the molecular basis and the biological significance of this interaction are unknown. In this study, we report that the regulatory subunit of PP2A (PP2AA) also interacts with the cytoplasmic tail of CTLA-4. Interestingly, TCR ligation induces tyrosine phosphorylation of PP2AA and its dissociation from CTLA-4 when coligated. The association between PP2AA and CTLA-4 involves a conserved three-lysine motif in the juxtamembrane portion of the cytoplasmic tail of CTLA-4. Mutations of these lysine residues prevent the binding of PP2AA and enhance the inhibition of IL-2 gene transcription by CTLA-4, indicating that PP2A represses CTLA-4 function. Our data imply that the lysine-rich motif in CTLA-4 may be used to identify small molecules that block its binding to PP2A and act as agonists for CTLA-4 function.

Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.