Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a costimulatory molecule that negatively regulates T-cell activation. Originally identified in murine CD8⁺ T cells, it has been found to be rapidly induced on human T cells. Furthermore, CTLA-4 is expressed on regulatory T cells. Clinically, targeting CTLA-4 has clinical utility in the treatment of melanoma. Whether the expression of CTLA-4 is differentially regulated in CD8⁺ vs CD4⁺ human T cells is unclear. Here, we analyzed CTLA-4 in normal human CD4⁺ and CD8⁺ T-cell subsets and show for the first time that CTLA-4 is expressed significantly higher in the CD4⁺ T cells than in CD8⁺ T cells. CTLA-4 is higher at the protein and the transcriptional levels in CD4⁺ T cells. This increase is due to the activation of the CTLA-4 promoter, which undergoes acetylation at the proximal promoter. Furthermore, we show that blocking CTLA-4 on CD4⁺ T cells permits greater proliferation in CD4⁺ vs CD8⁺ cells. These findings demonstrate a differential regulation of CTLA-4 on CD4⁺ and CD8⁺ T-cell subsets, which is likely important to the clinical efficacy for anti-CTLA-4 therapies. The findings hint to strategies to modulate CTLA-4 expression by targeting epigenetic transcription to alter the immune response.

CTLA-4 is a member of the costimulatory family, has homology to CD28, and binds the B7 family of ligands. Unlike CD28, CTLA-4 ligation transmits a negative signal in T cells. CTLA-4 expression, while inducible in most T cells, is expressed constitutively on T cells with a regulatory phenotype. The mechanism controlling CTLA-4 expression in human T cells is poorly characterized, thus we sought to better understand the mechanism of activation of the CTLA-4 gene. By cloning the 5′ upstream promoter and creating promoter-deletion reporter constructs, we show that the proximal promoter is critical for activating the CTLA-4 gene. Within this region, we identify a NFAT consensus sequence that binds NFAT with high affinity that differs from other NFAT sequences and does not recruit AP-1. Analysis of the chromatin proteins in the native CTLA-4 gene shows that this promoter region becomes associated with acetylated histones by chromatin immunoprecipitation assays. In addition, NFAT1 binds to the promoter of the CTLA-4 gene after stimulation by chromatin immunoprecipitation. The functional requirement of the NFAT site for CTLA-4 transcription was demonstrated by mutations in the NFAT site that abolished the activity of the promoter. Furthermore, inhibitors of NFAT suppressed CTLA-4 gene expression, indicating that NFAT plays a critical role in regulating the induction of the CTLA-4 gene in lymphocytes. The identification of NFAT as a critical regulator of the CTLA-4 gene suggests that targeting NFAT function may lead to novel approaches to modulate the CTLA-4 gene to control the immune response.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu+ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo. Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3+ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ. Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706–17. ©2018 AACR.

Purpose: Mycosis fungoides (MF) is a cutaneous T-cell lymphoma (CTCL) characterized by neoplastic skin-homing T cells. To better understand the immunopathogenesis of MF, we analyzed the functional ability of peripheral blood mononuclear cells (PBMC) from early and late MF/CTCL patients to express cytokine genes. In late stage MF/CTCL, patients were separated into those with blood involvement (+B) and without blood involvement (−B). Experimental Design: We analyzed T_H1 (interleukin 2 (IL-2), IFN-γ), T_H2 (IL-4, IL-5, IL-10, IL-13), and T_H17 (IL-17) cytokine gene expression from activated PBMCs from normal (n = 12), psoriasis (n = 6), early MF/CTCL (n = 11), and late MF/CTCL+B (n = 4) and MF/CTCL−B (n = 3) by quantitative real-time PCR. Results: PBMCs from early MF/CTCL and psoriasis showed higher induction of IL-2, IL-4, and IFN-γ genes than those from normal and late MF/CTCL−B and MF/CTCL+B (P < 0.05) in descending order. PBMCs from late MF/CTCL−B exhibited generally the highest level of IL-5, IL-10, IL-13, and IL-17 expression compared with the other groups. PBMCs from early MF/CTCL and late MF/CTCL−B had similarly elevated IL-13 and IL-17. Of all groups, PBMCs from late MF/CTCL+B had the lowest levels of IL-2 (P < 0.05), IL-4, IFN-γ, IL-13, and IL-17. Conclusions: The different pattern of cytokine gene expression suggests a change in immune function in MF/CTCL from early MF/CTCL to late MF/CTCL−B to late MF/CTCL+B. These stages are consistent with localized disease associated with an anti-tumor immune response and late MF/CTCL associated with a loss of immune function mediated by malignant T cells that share regulatory T cell–like properties.

The past two decades have brought impressive advancements in immune modulation, particularly with the advent of both cancer immunotherapy and biologic therapeutics for inflammatory conditions. However, the dynamic nature of the immune response often complicates the assessment of therapeutic outcomes. Innovative imaging technologies are designed to bridge this gap and allow non-invasive visualization of immune cell presence and/or function in real time. A variety of anatomical and molecular imaging modalities have been applied for this purpose, with each option providing specific advantages and drawbacks. Anatomical methods including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound provide sharp tissue resolution, which can be further enhanced with contrast agents, including super paramagnetic ions (for MRI) or nanobubbles (for ultrasound). Conjugation of the contrast material to an antibody allows for specific targeting of a cell population or protein of interest. Protein platforms including antibodies, cytokines, and receptor ligands are also popular choices as molecular imaging agents for positron emission tomography (PET), single-photon emission computerized tomography (SPECT), scintigraphy, and optical imaging. These tracers are tagged with either a radioisotope or fluorescent molecule for detection of the target. During the design process for immune-monitoring imaging tracers, it is important to consider any potential downstream physiologic impact. Antibodies may deplete the target cell population, trigger or inhibit receptor signaling, or neutralize the normal function(s) of soluble proteins. Alternatively, the use of cytokines or other ligands as tracers may stimulate their respective signaling pathways, even in low concentrations. As in vivo immune imaging is still in its infancy, this review aims to describe the modalities and immunologic targets that have thus far been explored, with the goal of promoting and guiding the future development and application of novel imaging technologies.