Intratumoral antigen signaling traps CD8+ T cells to confine exhaustion to the tumor site.

Immunotherapy advances have been hindered by difficulties in tracking the behaviors of lymphocytes after antigen signaling. Here, we assessed the behavior of T cells active within tumors through the development of the antigen receptor signaling reporter (AgRSR) mouse, fate-mapping lymphocytes responding to antigens at specific times and locations. Contrary to reports describing the ready egress of T cells out of the tumor, we find that intratumoral antigen signaling traps CD8+ T cells in the tumor. These clonal populations expand and become increasingly exhausted over time. By contrast, antigen-signaled regulatory T cell (Treg) clonal populations readily recirculate out of the tumor. Consequently, intratumoral antigen signaling acts as a gatekeeper to compartmentalize CD8+ T cell responses, even within the same clonotype, thus enabling exhausted T cells to remain confined to a specific tumor tissue site.

GS-TCGA: Gene Set-Based Analysis of The Cancer Genome Atlas.

Most tools for analyzing large gene expression datasets, including The Cancer Genome Atlas (TCGA), have focused on analyzing the expression of individual genes or inference of the abundance of specific cell types from whole transcriptome information. While these methods provide useful insights, they can overlook crucial process-based information that may enhance our understanding of cancer biology. In this study, we describe three novel tools incorporated into an online resource; gene set-based analysis of The Cancer Genome Atlas (GS-TCGA). GS-TCGA is designed to enable user-friendly exploration of TCGA data using gene set-based analysis, leveraging gene sets from the Molecular Signatures Database. GS-TCGA includes three unique tools: GS-Surv determines the association between the expression of gene sets and survival in human cancers. Co-correlative gene set enrichment analysis (CC-GSEA) utilizes interpatient heterogeneity in cancer gene expression to infer functions of specific genes based on GSEA of coregulated genes in TCGA. GS-Corr utilizes interpatient heterogeneity in cancer gene expression profiles to identify genes coregulated with the expression of specific gene sets in TCGA. Users are also able to upload custom gene sets for analysis with each tool. These tools empower researchers to perform survival analysis linked to gene set expression, explore the functional implications of gene coexpression, and identify potential gene regulatory mechanisms.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Intracellular K+ Limits T-cell Exhaustion and Preserves Antitumor Function.

T cells are often compromised within cancers, allowing disease progression. We previously found that intratumoral elevations in extracellular K+, related to ongoing cell death, constrained CD8+ T-cell Akt-mTOR signaling and effector function. To alleviate K+-mediated T-cell dysfunction, we pursued genetic means to lower intracellular K+. CD8+ T cells robustly and dynamically express the Na+/K+ ATPase, among other K+ transporters. CRISPR-Cas9-mediated disruption of the Atp1a1 locus lowered intracellular K+ and elevated the resting membrane potential (i.e., Vm, Ψ). Despite compromised Ca2+ influx, Atp1a1-deficient T cells harbored tonic hyperactivity in multiple signal transduction cascades, along with a phenotype of exhaustion in mouse and human CD8+ T cells. Provision of exogenous K+ restored intracellular levels in Atp1a1-deficient T cells and prevented damaging levels of reactive oxygen species (ROS), and both antioxidant treatment and exogenous K+ prevented Atp1a1-deficient T-cell exhaustion in vitro. T cells lacking Atp1a1 had compromised persistence and antitumor activity in a syngeneic model of orthotopic murine melanoma. Translational application of these findings will require balancing the beneficial aspects of intracellular K+ with the ROS-dependent nature of T-cell effector function. See related Spotlight by Banuelos and Borges da Silva, p. 6.

Acquisition of suppressive function by conventional T cells limits antitumor immunity upon Treg depletion.

Regulatory T (Treg) cells contribute to immune homeostasis but suppress immune responses to cancer. Strategies to disrupt Treg cell-mediated cancer immunosuppression have been met with limited clinical success, but the underlying mechanisms for treatment failure are poorly understood. By modeling Treg cell-targeted immunotherapy in mice, we find that CD4+ Foxp3- conventional T (Tconv) cells acquire suppressive function upon depletion of Foxp3+ Treg cells, limiting therapeutic efficacy. Foxp3- Tconv cells within tumors adopt a Treg cell-like transcriptional profile upon ablation of Treg cells and acquire the ability to suppress T cell activation and proliferation ex vivo. Suppressive activity is enriched among CD4+ Tconv cells marked by expression of C-C motif receptor 8 (CCR8), which are found in mouse and human tumors. Upon Treg cell depletion, CCR8+ Tconv cells undergo systemic and intratumoral activation and expansion, and mediate IL-10-dependent suppression of antitumor immunity. Consequently, conditional deletion of Il10 within T cells augments antitumor immunity upon Treg cell depletion in mice, and antibody blockade of IL-10 signaling synergizes with Treg cell depletion to overcome treatment resistance. These findings reveal a secondary layer of immunosuppression by Tconv cells released upon therapeutic Treg cell depletion and suggest that broader consideration of suppressive function within the T cell lineage is required for development of effective Treg cell-targeted therapies.

Intracellular K + limits T cell exhaustion and preserves antitumor function.

The cancer-killing activity of T cells is often compromised within tumors, allowing disease progression. We previously found that intratumoral elevations in extracellular K + related to ongoing cell death constrained CD8 + T cell Akt-mTOR signaling and effector function (1,2). To alleviate K + mediated T cell suppression, we pursued genetic means to lower intracellular K + . Transcriptomic analysis of CD8 + T cells demonstrated the Na + /K + ATPase to be robustly and dynamically expressed. CRISPR-Cas9 mediated deletion of the catalytic alpha subunit of the Na + /K + ATPase lowered intracellular K + but produced tonic hyperactivity in multiple signal transduction cascades along with the acquisition of co-inhibitory receptors and terminal differentiation in mouse and human CD8 + T cells. Mechanistically, Na + /K + ATPase disruption led to ROS accumulation due to depletion of intracellular K + in T cells. Antioxidant treatment or high K + media prevented Atp1a1 deficient T cells from exhausted T (T Ex ) cell formation. Consistent with transcriptional and proteomic data suggesting a T Ex cell phenotype, T cells lacking Atp1a1 had compromised persistence and antitumor activity in a syngeneic model of orthotopic murine melanoma. Translational application of these findings will include efforts to lower intracellular K + while limiting ROS accumulation within tumor specific T cells. Synopsis: High extracellular K + (↑[K + ] e ) is found within tumors and suppresses T cell effector function. Collier et al. find that deletion of the Na + /K + ATPase in T cells lowers intracellular K + and promotes ROS accumulation, tonic signal transduction and T cell exhaustion owing to ROS accumulation. Engineering T cell ion transport is an important consideration for cancer immunotherapy.

High-Dimensional Single-Cell Profiling of Tumor-Infiltrating CD4+ Regulatory T Cells.

CD4+ T regulatory cells (Tregs) are a specialized subset of T lymphocytes, which promote immune homeostasis and tumor immunosuppression by restricting effector T cell immune responses. The characterization of context-specific Treg phenotypic heterogeneity is pivotal to determine their potential contributions to diseases. In the recent years, high-dimensional single-cell technologies, such as single-cell RNA sequencing, mass cytometry, or polychromatic flow cytometry, have played a central role in elucidating the heterogeneity of the Treg compartment at the cellular and molecular levels. Here we describe an example of high-dimensional flow cytometry analysis capable of defining an effector Treg subpopulation that positively correlates with cancer progression. Moreover, we provide a workflow template of high-dimensional single-cell analysis that is readily applicable to any leukocyte subpopulation.

IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis.

Natural killer (NK) cells are critical to immune surveillance against infections and cancer. Their role in immune surveillance requires that NK cells are present within tissues in a quiescent state. Mechanisms by which NK cells remain quiescent in tissues are incompletely elucidated. The transcriptional repressor BACH2 plays a critical role within the adaptive immune system, but its function within innate lymphocytes has been unclear. Here, we show that BACH2 acts as an intrinsic negative regulator of NK cell maturation and function. BACH2 is expressed within developing and mature NK cells and promotes the maintenance of immature NK cells by restricting their maturation in the presence of weak stimulatory signals. Loss of BACH2 within NK cells results in accumulation of activated NK cells with unrestrained cytotoxic function within tissues, which mediate augmented immune surveillance to pulmonary cancer metastasis. These findings establish a critical function of BACH2 as a global negative regulator of innate cytotoxic function and tumor immune surveillance by NK cells.

Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

BACKGROUND & AIMS: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). METHODS: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. RESULTS: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. CONCLUSIONS: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. LAY SUMMARY: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

TCR-induced FOXP3 expression by CD8+ T cells impairs their anti-tumor activity.

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-γ, IFN-α, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function.

CD4+ regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8-/- mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which were abolished in Ccr8-/- mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8+ Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

A cell-based bioluminescence assay reveals dose-dependent and contextual repression of AP-1-driven gene expression by BACH2.

Whereas effector CD4+ and CD8+ T cells promote immune activation and can drive clearance of infections and cancer, CD4+ regulatory T (Treg) cells suppress their function, contributing to both immune homeostasis and cancer immunosuppression. The transcription factor BACH2 functions as a pervasive regulator of T cell differentiation, promoting development of CD4+ Treg cells and suppressing the effector functions of multiple effector T cell (Teff) lineages. Here, we report the development of a stable cell-based bioluminescence assay of the transcription factor activity of BACH2. Tetracycline-inducible BACH2 expression resulted in suppression of phorbol 12-myristate 13-acetate (PMA)/ionomycin-driven activation of a luciferase reporter containing BACH2/AP-1 target sequences from the mouse Ifng + 18k enhancer. BACH2 expression repressed the luciferase signal in a dose-dependent manner but this activity was abolished at high levels of AP-1 signalling, suggesting contextual regulation of AP-1 driven gene expression by BACH2. Finally, using the reporter assay developed, we find that the histone deacetylase 3 (HDAC3)-selective inhibitor, RGFP966, inhibits BACH2-mediated repression of signal-driven luciferase expression. In addition to enabling mechanistic studies, this cell-based reporter may enable identification of small molecule agonists or antagonists of BACH2 function for drug development.

A distal enhancer at risk locus 11q13.5 promotes suppression of colitis by Treg cells.

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.

BACH2 drives quiescence and maintenance of resting Treg cells to promote homeostasis and cancer immunosuppression.

Regulatory T (Treg) cell populations are composed of functionally quiescent resting Treg (rTreg) cells which differentiate into activated Treg (aTreg) cells upon antigen stimulation. How rTreg cells remain quiescent despite chronic exposure to cognate self- and foreign antigens is unclear. The transcription factor BACH2 is critical for early Treg lineage specification, but its function following lineage commitment is unresolved. Here, we show that BACH2 is repurposed following Treg lineage commitment and promotes the quiescence and long-term maintenance of rTreg cells. Bach2 is highly expressed in rTreg cells but is down-regulated in aTreg cells and during inflammation. In rTreg cells, BACH2 binds to enhancers of genes involved in aTreg differentiation and represses their TCR-driven induction by competing with AP-1 factors for DNA binding. This function promotes rTreg cell quiescence and long-term maintenance and is required for immune homeostasis and durable immunosuppression in cancer. Thus, BACH2 supports a "division of labor" between quiescent rTreg cells and their activated progeny in Treg maintenance and function, respectively.

IRF4 instructs effector Treg differentiation and immune suppression in human cancer.

The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type.

Genome-Wide Measurement and Computational Analysis of Transcription Factor Binding and Chromatin Accessibility in Lymphocytes.

Cells of the adaptive immune system, including CD4+ and CD8+ T cells, as well as B cells, possess the ability to undergo dynamic changes in population size, differentiation state, and function to counteract diverse and temporally stochastic threats from the external environment. To achieve this, lymphocytes must be able to rapidly control their gene-expression programs in a cell-type-specific manner and in response to extrinsic signals. Such capacity is provided by transcription factors (TFs), which bind to the available repertoire of regulatory DNA elements in distinct lymphocyte subsets to program cell-type-specific gene expression. Here we provide a set of protocols that utilize massively parallel sequencing-based approaches to map genome-wide TF-binding sites and accessible chromatin, with consideration of the unique aspects and technical issues facing their application to lymphocytes. We show how to computationally validate and analyze aligned data to map differentially enriched/accessible sites, identify enriched DNA sequence motifs, and detect the position of nucleosomes adjacent to accessible DNA elements. These techniques, when applied to immune cells, can enhance our understanding of how gene-expression programs are controlled within lymphocytes to coordinate immune function in homeostasis and disease. © 2019 by John Wiley & Sons, Inc.

Regulatory T cells in cancer: where are we now?

There have been substantial strides forward in our understanding of the contribution of regulatory T (Treg) cells to cancer immunosuppression. In this issue, we present a series of papers highlighting emerging themes on this topic relevant not only to our understanding of the fundamental biology of tumour immunosuppression but also to the design of new immunotherapeutic approaches. The substantially shared biology of CD4+ conventional T (Tconv) and Treg cells necessitates a detailed understanding of the potentially opposing functional consequences that immunotherapies will have on Treg and Tconv cells, a prominent example being the potential for Treg-mediated hyperprogressive disease following anti-PD-1 therapy. Such understanding will aid patient stratification and the rational design of combination therapies. It is also becoming clear, however, that Treg cells within tumours exhibit distinct biological features to both Tconv cells and Treg cells in other tissues. These distinct features provide the opportunity for development of targeted immunotherapies with greater efficacy and reduced potential for inducing systemic toxicity.