Non-canonical function of histone methyltransferase G9a in the translational regulation of chronic inflammation.

We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications. Using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m6A) RNA methyltransferase METTL3, to co-upregulate expression of certain m6A-modified mRNAs that encode immune-checkpoint and anti-inflammatory proteins. Mechanistically, G9a promotes m6A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m6A translation regulatory axis, our translatome proteomics approach identified numerous "G9a-translated" proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases.

Intricacies of TGF-ß signaling in Treg and Th17 cell biology.

Balanced immunity is pivotal for health and homeostasis. CD4+ helper T (Th) cells are central to the balance between immune tolerance and immune rejection. Th cells adopt distinct functions to maintain tolerance and clear pathogens. Dysregulation of Th cell function often leads to maladies, including autoimmunity, inflammatory disease, cancer, and infection. Regulatory T (Treg) and Th17 cells are critical Th cell types involved in immune tolerance, homeostasis, pathogenicity, and pathogen clearance. It is therefore critical to understand how Treg and Th17 cells are regulated in health and disease. Cytokines are instrumental in directing Treg and Th17 cell function. The evolutionarily conserved TGF-ß (transforming growth factor-ß) cytokine superfamily is of particular interest because it is central to the biology of both Treg cells that are predominantly immunosuppressive and Th17 cells that can be proinflammatory, pathogenic, and immune regulatory. How TGF-ß superfamily members and their intricate signaling pathways regulate Treg and Th17 cell function is a question that has been intensely investigated for two decades. Here, we introduce the fundamental biology of TGF-ß superfamily signaling, Treg cells, and Th17 cells and discuss in detail how the TGF-ß superfamily contributes to Treg and Th17 cell biology through complex yet ordered and cooperative signaling networks.

Caspase-8 contributes to an immuno-hot microenvironment by promoting phagocytosis via an ecto-calreticulin-dependent mechanism.

BACKGROUND: Caspase-8 (Casp8) acts as an initiator in cell apoptosis signaling. However, the role of Casp8 in tuning the tumor immune microenvironment remains controversial due to the complicated crosstalk between immune-tolerogenic apoptotic cell death and immunogenic cell death cascades. METHODS: The Cancer Genome Atlas (TCGA) and publicly accessible immune checkpoint blockade (ICB)-treated cohorts were used to investigate the clinical relevance of Casp8. A tumor-bearing mouse model was used to characterize changes in the tumor microenvironment and to explore the efficacy of ICB treatment under Casp8 knockout conditions. RESULTS: By exploring TCGA datasets, we showed that the expression level of Casp8 was associated with an immuno-hot microenvironment across various solid tumor types. Casp8 deficiency leads to decreased CD8+ T cell infiltration and resistance to anti-PD-L1 therapy in a mouse model. Mechanistically, Casp8 deficiency or pharmacological disruption results in impaired ecto-calreticulin transition in tumor cells, which in turn hampers antigen presentation in draining lymph nodes. Furthermore, radiotherapy restored sensitivity to anti-PD-L1 treatment via elevated calreticulin surface expression. CONCLUSIONS: Our data revealed a causative role of Casp8 in modulating the immunogenicity of tumor cells and responsiveness to ICB immunotherapies and proposed radiotherapy as a salvage approach to overcome Casp8 deficiency-mediated ICB resistance.

CD200+ cytotoxic T lymphocytes in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy.

Anti-PD-1/PD-L1 therapy, either by anti-PD-1 antibody or anti-PD-L1 antibody, has efficacy by reinvigorating tumor-infiltrating CD8+ T cells in a subset of patients with cancer, but it has unequal effects on heterogeneous CD8+ T cell populations. Hence, the subset crucial to efficacious PD-1 blockade therapy remains elusive. Here, we found an increase in tumor-infiltrating CD200+ cytotoxic T lymphocytes (CTLs) upon PD-1/PD-L1 blockade, with higher proportions of CD200+ T cells positively related to a favorable clinical outcome to anti-PD-1/PD-L1 therapy in three independent cohorts of patients with cancer. Using multiple mouse tumor models, we demonstrated that CD200+ CTLs are essential for efficacious anti-PD-L1 therapy. Mechanistically, we observed a unique chromatin landscape in CD200+ CTLs and found that these cells are enriched for tumor antigen-specific CTLs and have antitumor effector functions. Coinoculation of CD200+ CTLs with tumor cells led to robust tumor regression in two transplanted mouse models. Clinically, we found that infiltration of CD200+ CTLs into tumors could predict immunotherapy efficacy in six patient cohorts. Together, our findings reveal that CD200+ CTLs in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy and could serve as a predictor of successful immunotherapy in the clinic.

Ribosomal protein RPL11 haploinsufficiency causes anemia in mice via activation of the RP-MDM2-p53 pathway.

Recent discovery of the ribosomal protein (RP) RPL11 interacting with and inhibiting the E3 ubiquitin ligase function of MDM2 established the RP-MDM2-p53 signaling pathway, which is linked to biological events, including ribosomal biogenesis, nutrient availability, and metabolic homeostasis. Mutations in RPs lead to a diverse array of phenotypes known as ribosomopathies in which the role of p53 is implicated. Here, we generated conditional RPL11-deletion mice to investigate in vivo effects of impaired RP expression and its functional connection with p53. While deletion of one Rpl11 allele in germ cells results in embryonic lethality, deletion of one Rpl11 allele in adult mice does not affect viability but leads to acute anemia. Mechanistically, we found RPL11 haploinsufficiency activates p53 in hematopoietic tissues and impedes erythroid precursor differentiation, resulting in insufficient red blood cell development. We demonstrated that reducing p53 dosage by deleting one p53 allele rescues RPL11 haploinsufficiency-induced inhibition of erythropoietic precursor differentiation and restores normal red blood cell levels in mice. Furthermore, blocking the RP-MDM2-p53 pathway by introducing an RP-binding mutation in MDM2 prevents RPL11 haploinsufficiency-caused p53 activation and rescues the anemia in mice. Together, these findings demonstrate that the RP-MDM2-p53 pathway is a critical checkpoint for RP homeostasis and that p53-dependent cell cycle arrest of erythroid precursors is the molecular basis for the anemia phenotype commonly associated with RP deficiency.

Tumor-induced erythroid precursor-differentiated myeloid cells mediate immunosuppression and curtail anti-PD-1/PD-L1 treatment efficacy.

Despite the unprecedented success of immune checkpoint inhibitors (ICIs) as anti-cancer therapy, it remains a prevailing clinical need to identify additional mechanisms underlying ICI therapeutic efficacy and potential drug resistance. Here, using lineage tracking in cancer patients and tumor-bearing mice, we demonstrate that erythroid progenitor cells lose their developmental potential and switch to the myeloid lineage. Single-cell transcriptome analyses reveal that, notwithstanding quantitative differences in erythroid gene expression, erythroid differentiated myeloid cells (EDMCs) are transcriptionally indistinguishable from their myeloid-originated counterparts. EDMCs possess multifaceted machinery to curtail T cell-mediated anti-tumor responses. Consequently, EDMC content within tumor tissues is negatively associated with T cell inflammation for the majority of solid cancers; moreover, EDMC enrichment, in accordance with anemia manifestation, is predictive of poor prognosis in various cohorts of patients undergoing ICI therapy. Together, our findings reveal a feedforward mechanism by which tumors exploit anemia-triggered erythropoiesis for myeloid transdifferentiation and immunosuppression.

SKI Expression Suppresses Pathogenic Th17 Cell Response and Mitigates Experimental Autoimmune Encephalomyelitis.

Pathogenic Th17 cells are critically involved in many autoimmune diseases, while non-pathogenic Th17 cells are more immune regulatory. Understanding the mechanisms of the induction and maintenance of pathogenic Th17 cells will benefit the development of therapeutic treatments of related diseases. We have shown that the transforming growth factor-ß (TGFß) induced SKI degradation and dissociation from Smad4 complex is a prerequisite for TGFß-induced Th17 cell differentiation. However, it is unclear whether and how SKI regulates pathogenic Th17 differentiation, which does not require TGFß cytokine. Here we showed that SKI expression was downregulated during pathogenic Th17 cell differentiation and the ectopic expression of SKI abrogated the differentiation of pathogenic Th17 cells. Functionally, using a knock-in mouse model, we found ectopic SKI expression specifically in T cells prevented myelin oligodendrocyte glycoprotein peptide (MOG33-55) induced experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. We further revealed that induced SKI expression in already differentiated pathogenic Th17 cells reduced the maintenance of Th17 program and ameliorated EAE in an adoptive T cell transfer model. Therefore, our study provides valuable insights of targeting SKI to modulate pathogenic Th17 cell function and treat Th17-related diseases.

AIM2 in regulatory T cells restrains autoimmune diseases.

The inflammasome initiates innate defence and inflammatory responses by activating caspase-1 and pyroptotic cell death in myeloid cells1,2. It consists of an innate immune receptor/sensor, pro-caspase-1, and a common adaptor molecule, ASC. Consistent with their pro-inflammatory function, caspase-1, ASC and the inflammasome component NLRP3 exacerbate autoimmunity during experimental autoimmune encephalomyelitis by enhancing the secretion of IL-1ß and IL-18 in myeloid cells3-6. Here we show that the DNA-binding inflammasome receptor AIM27-10 has a T cell-intrinsic and inflammasome-independent role in the function of T regulatory (Treg) cells. AIM2 is highly expressed by both human and mouse Treg cells, is induced by TGFß, and its promoter is occupied by transcription factors that are associated with Treg cells such as RUNX1, ETS1, BCL11B and CREB. RNA sequencing, biochemical and metabolic analyses demonstrated that AIM2 attenuates AKT phosphorylation, mTOR and MYC signalling, and glycolysis, but promotes oxidative phosphorylation of lipids in Treg cells. Mechanistically, AIM2 interacts with the RACK1-PP2A phosphatase complex to restrain AKT phosphorylation. Lineage-tracing analysis demonstrates that AIM2 promotes the stability of Treg cells during inflammation. Although AIM2 is generally accepted as an inflammasome effector in myeloid cells, our results demonstrate a T cell-intrinsic role of AIM2 in restraining autoimmunity by reducing AKT-mTOR signalling and altering immune metabolism to enhance the stability of Treg cells.

The TGF-ß superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation.

Th17 cells are known to exert pathogenic and non-pathogenic functions. Although the cytokine transforming growth factor ß1 (TGF-ß1) is instrumental for Th17 cell differentiation, it is dispensable for generation of pathogenic Th17 cells. Here, we examined the T cell-intrinsic role of Activin-A, a TGF-ß superfamily member closely related to TGF-ß1, in pathogenic Th17 cell differentiation. Activin-A expression was increased in individuals with relapsing-remitting multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. Stimulation with interleukin-6 and Activin-A induced a molecular program that mirrored that of pathogenic Th17 cells and was inhibited by blocking Activin-A signaling. Genetic disruption of Activin-A and its receptor ALK4 in T cells impaired pathogenic Th17 cell differentiation in vitro and in vivo. Mechanistically, extracellular-signal-regulated kinase (ERK) phosphorylation, which was essential for pathogenic Th17 cell differentiation, was suppressed by TGF-ß1-ALK5 but not Activin-A-ALK4 signaling. Thus, Activin-A drives pathogenic Th17 cell differentiation, implicating the Activin-A-ALK4-ERK axis as a therapeutic target for Th17 cell-related diseases.

Radiation-induced eosinophils improve cytotoxic T lymphocyte recruitment and response to immunotherapy.

The efficacy of cancer immunotherapy is dictated by CD8+ T cell infiltration and the nature of the tumor microenvironment (TME). By inflaming the TME to favor CD8+ T cell immunity, radiation is now widely considered as a neoadjuvant for immunomodulation. Here, we observed that local irradiation enhances the infiltration of intratumoral eosinophils, and depletion of eosinophil dampens CD8+ T cell infiltration and diminishes the anti-tumor effectiveness of radiation. Retrospectively, we identified a strong correlation between eosinophilia and survival benefit in radiation-treated cancer patients. Experimentally, we further show that radiation enhances the intratumoral infiltration of adoptive transferred T cells therapy, bolstering eosinophils by intravenous interleukin-5 administration promotes the efficacy of radiation-induced abscopal effect. Together, these results suggest that eosinophil mobilization can be considered as a mechanistically relevant biomarker for predicting the effectiveness of pre-immunotherapy radiation, as well as a new strategy to enhance T cell-mediated immunotherapy against cancers.

Mutational burden and chromosomal aneuploidy synergistically predict survival from radiotherapy in non-small cell lung cancer.

Therapeutic radiation can result in substantially different survival outcomes for patients with non-small cell lung cancer (NSCLC). Measures for identification of patients who can benefit most throughout radiotherapy remain limited. In this retrospective study, survival analysis was performed based on a discovery cohort from TCGA and a validation cohort from three independent hospitals. Tumor mutational burden (TMB) and chromosomal aneuploidy (ANE) were derived from the whole exome sequencing (WES) data from treatment-naïve tumors. Integrated risk scores were derived from TMB and ANE by a multivariate Cox proportional hazards model. TCGA reveal that TMB and ANE are associated positively and negatively, respectively, with survival throughout radiotherapy. Additionally, the synergistically predictive significance of these two genomic alterations, in differing responders and non-responders to radiotherapy is identified. These biomarkers may have clinical potential to improve personalized treatment management by rationally identifying highly likely responders to therapeutic radiation in patients with NSCLC.

The SKI proto-oncogene restrains the resident CD103+CD8+ T cell response in viral clearance.

Acute viral infection causes illness and death. In addition, an infection often results in increased susceptibility to a secondary infection, but the mechanisms behind this susceptibility are poorly understood. Since its initial identification as a marker for resident memory CD8+ T cells in barrier tissues, the function and regulation of CD103 integrin (encoded by ITGAE gene) have been extensively investigated. Nonetheless, the function and regulation of the resident CD103+CD8+ T cell response to acute viral infection remain unclear. Although TGFß signaling is essential for CD103 expression, the precise molecular mechanism behind this regulation is elusive. Here, we reveal a TGFß-SKI-Smad4 pathway that critically and specifically directs resident CD103+CD8+ T cell generation for protective immunity against primary and secondary viral infection. We found that resident CD103+CD8+ T cells are abundant in both lymphoid and nonlymphoid tissues from uninfected mice. CD103 acts as a costimulation signal to produce an optimal antigenic CD8+ T cell response to acute viral infection. There is a reduction in resident CD103+CD8+ T cells following primary infection that results in increased susceptibility of the host to secondary infection. Intriguingly, CD103 expression inversely and specifically correlates with SKI proto-oncogene (SKI) expression but not R-Smad2/3 activation. Ectopic expression of SKI restricts CD103 expression in CD8+ T cells in vitro and in vivo to hamper viral clearance. Mechanistically, SKI is recruited to the Itgae loci to directly suppress CD103 transcription by regulating histone acetylation in a Smad4-dependent manner. Our study therefore reveals that resident CD103+CD8+ T cells dictate protective immunity during primary and secondary infection. Interfering with SKI function may amplify the resident CD103+CD8+ T cell response to promote protective immunity.

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics.

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

DCAF1 regulates Treg senescence via the ROS axis during immunological aging.

As a hallmark of immunological aging, low-grade, chronic inflammation with accumulation of effector memory T cells contributes to increased susceptibility to many aging-related diseases. While the proinflammatory state of aged T cells indicates a dysregulation of immune homeostasis, whether and how aging drives regulatory T cell (Treg) aging and alters Treg function are not fully understood owing to a lack of specific aging markers. Here, by a combination of cellular, molecular, and bioinformatic approaches, we discovered that Tregs senesce more severely than conventional T (Tconv) cells during aging. We found that Tregs from aged mice were less efficient than young Tregs in suppressing Tconv cell function in an inflammatory bowel disease model and in preventing Tconv cell aging in an irradiation-induced aging model. Furthermore, we revealed that DDB1- and CUL4-associated factor 1 (DCAF1) was downregulated in aged Tregs and was critical to restrain Treg aging via reactive oxygen species (ROS) regulated by glutathione-S-transferase P (GSTP1). Importantly, interfering with GSTP1 and ROS pathways reinvigorated the proliferation and function of aged Tregs. Therefore, our studies uncover an important role of the DCAF1/GSTP1/ROS axis in Treg senescence, which leads to uncontrolled inflammation and immunological aging.

Inhibition of Cdk8/Cdk19 Activity Promotes Treg Cell Differentiation and Suppresses Autoimmune Diseases.

Foxp3 expressing regulatory T (Treg) cells, as the central negative regulator of adaptive immune system, are essential to suppress immune response and maintain immune homeostasis. However, the function of Treg cells is frequently compromised in autoimmunity and hyper-activated in infections and tumor microenvironments. Thus, manipulating Treg cells becomes a promising therapeutic strategy for treating various diseases. Here we reported that inhibition of Cdk8/Cdk19 activity by small molecule inhibitors CCT251921 or Senexin A greatly promoted the differentiation of Treg cells and the expression of Treg signature genes, such as Foxp3, CTLA4, PD-1, and GITR. Mechanistically, we found that the augmented Treg cell differentiation was due to sensitized TGF-ß signaling by Cdk8/Cdk19 inhibition, which was associated with attenuation of IFN-γ-Stat1 signaling and enhancement of phosphorylated Smad2/3. Importantly, treatment with Cdk8/Cdk19 inhibitor CCT251921 significantly increased Treg population and ameliorated autoimmune symptoms in an experimental autoimmune encephalomyelitis (EAE) model. Taken together, our study reveals a novel role of Cdk8/Cdk19 in Treg cell differentiation and provides a potential target for Treg cell based therapeutics.

SKI and SMAD4 are essential for IL-21-induced Th17 differentiation.

Th17 cells are essential for the pathogenesis of inflammatory and autoimmune diseases. In the presence of TGF-ß, the differentiation of Th17 cells can be induced by inflammatory cytokines, especially IL-6, which is mainly produced by antigen presenting cells (APCs); or IL-21, which is derived from T cells. IL-21 is required for IL-6-induced Th17 cell differentiation. However, the key regulators and underlying mechanisms for IL-21-induced Th17 differentiation is still elusive. Here we show that SMAD4 is a key regulator in IL-21-induced Th17 differentiation. SMAD4 deficient naïve T cells can differentiate into Th17 cells in the absence of TGF-ß signaling, and these Th17 cells are pathogenic during EAE. SMAD4 represses Rorc mRNA transcription to constrain IL-21-induced Th17 differentiation in the absence of TGF-ß signaling. While in the presence of TGF-ß, SMAD4 losses its suppressive ability due to the degradation of SKI. Mutation of Y429A or A432E on SMAD4 disrupts the interaction of SKI from SMAD4 and eliminates SMAD4 mediated suppression of Th17 differentiation. SMAD4 is indispensable for SKI binding to Rorc promoter region to regulate Th17 differentiation. Moreover, activin can induce Th17 differentiation in combination with IL-21, and the process is also subjected to the control of SKI and SMAD4. This study therefore elucidates critical mechanism for IL-21-induced Th17 differentiation to indicate SKI and SMAD4 as potential therapeutic targets for treating autoimmune diseases.

Targeting EZH2 histone methyltransferase activity alleviates experimental intestinal inflammation.

Enhancer of zeste homolog 2 (EZH2)-mediated trimethylation of histone 3 lysine 27 (H3K27Me3) is critical for immune regulation. However, evidence is lacking to address the effect of EZH2 enzyme's activity on intestinal immune responses during inflammatory bowel disease (IBD). Here we report that suppressing EZH2 activity ameliorates experimental intestinal inflammation and delayed the onset of colitis-associated cancer. In addition, we identified an increased number of functional MDSCs in the colons, which are essential for EZH2 inhibitor activity. Moreover, inhibition of EZH2 activity promotes the generation of MDSCs from hematopoietic progenitor cells in vitro, demonstrating a previously unappreciated role for EZH2 in the development of MDSCs. Together, these findings suggest the feasibility of EZH2 inhibitor clinical trials for the control of IBD. In addition, this study identifies MDSC-promoting effects of EZH2 inhibitors that may be undesirable in other therapeutic contexts and should be addressed in a clinical trial setting.

EZH2 Inhibitor GSK126 Suppresses Antitumor Immunity by Driving Production of Myeloid-Derived Suppressor Cells.

Enhancer of zeste homolog (EZH2) is a key epigenetic regulator of gene expression and is frequently overexpressed in various cancer types, suggesting a role in oncogenesis. The therapeutic potential of EZH2 inhibitors is currently being explored, but their effect on antitumor immunity is largely unknown. Here we report that suppressing EZH2 activity using EZH2 inhibitor GSK126 resulted in increased numbers of myeloid-derived suppressor cells (MDSC) and fewer CD4+ and IFNγ+CD8+ T cells, which are involved in antitumor immunity. Addition of a neutralizing antibody against the myeloid differentiation antigen GR-1 or gemcitabine/5-fluorouracil-depleted MDSCs alleviated MDSC-mediated immunosuppression and increased CD4+ and CD8+ T-cell tumor infiltration and GSK126 therapeutic efficacy. Mechanistically, we identified a novel pathway of MDSC production in cancer in which EZH2 inhibition directs myeloid differentiation from primitive hematopoietic progenitor cells. These findings suggest that modulating the tumor immune microenvironment may improve the efficacy of EZH2 inhibitors. SIGNIFICANCE: This study uncovers a potential mechanism behind disappointing results of a phase I clinical trial of EZH2 inhibitor GSK126 and identifies a translatable combinational strategy to overcome it.

Intracellular Activation of Complement C3 Leads to PD-L1 Antibody Treatment Resistance by Modulating Tumor-Associated Macrophages.

Complement aids in the construction of an immunosuppressive tumor microenvironment. Tumor cell-derived C3 has been previously reported, but whether and how it acts on antitumor immunity remains to be elucidated. Here, we describe a mechanism for tumor cell-derived C3 in suppressing antitumor immunity. Tumor cell-derived C3 was activated intracellularly, which results in generation of C3a. C3a modulated tumor-associated macrophages via C3a-C3aR-PI3Kγ signaling, thereby repressing antitumor immunity. Deletion of C3 in tumor cells that had high C3 expression enhanced efficacy of anti-PD-L1 treatment. Collectively, our results suggest tumor cell-derived C3 may be a useful target for cancer immunotherapy and that targeting C3 in tumor cells may enhance antitumor immunity.