Supplementary Figure 1. Disease status of patients in the study compared with those of broader melanoma populations.

The frequency of peripheral Tregs in tumor-bearing αCCR8-treated mice and the effects of αCCR8 on ex vivo tumor CD8+ and CD4+ T cells.

File contains the supplementary Material and Methods

CCR8 mAb treatment mediates potent antitumor activity, suppressing MC38 tumor growth and improving long-term survival.

Expression of Lm-AH1 vaccine construct.

AbstractCCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes–based immunotherapy. Anti-CCR8 mAb therapy synergized with L. monocytogenes–based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies.Significance: Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. Cancer Res; 78(18); 5340–8. ©2018 AACR.

The interaction between T cells and the central nervous system (CNS) in homeostasis and injury has been recognized being both pathogenic (CD4+ T-helper 1 - Th1, Th17 and γδT) and ameliorative (Th2 and regulatory T cells - Tregs). However, in-depth studies aimed to elucidate the precise in the aged microenvironment and the dichotomous role of Tregs have just begun and many aspects remain unclear. This is due, not only to a mutual dependency and reciprocal causation of alterations and diseases between the nervous and T cell immune systems, but also to an inconsistent aging of the two systems, which dynamically changes with CNS injury/recovery and/or aging process. Cellular immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution - sources of chronic inflammation in the elderly (termed inflammaging), potentially induces an acceleration of brain aging and memory loss. In turn, aging of the brain via neuro-endocrine-immune network drives total body systemic aging, including that of the immune system. Therefore, immunotherapeutics including vaccination and “protective autoimmunity” provide promising means to rejuvenate neuro-inflammatory disorders and repair CNS acute injury and chronic neuro-degeneration. We review the current understanding and recent discoveries linking the aging immune system with CNS injury and neuro-degeneration. Additionally, we discuss potential recovery and rejuvenation strategies, focusing on targeting the aging T cell immune system in an effort to alleviate acute brain injury and chronic neuro-degeneration during aging, via the “thymus-inflammaging-neurodegeneration axis”.

Lymphatic metastasis is a primary cause of gastric cancer-related death, yet factors governing tumor cell lymphatic metastasis have not been fully elucidated. Little is known about the contributions of long noncoding RNAs (lncRNAs) to lymphatic metastasis in gastric cancer. Differentially expressional lncRNAs between metastatic lymph node tissues and normal lymph node tissues were identified and validated by microarray and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Our results found that the expression level of C21orF96 was over-expressed in positive lymph node tissues and gastric cancer tissues. We evaluated the altered expressions of C21orF96 in gastric cancer tissues comparing to adjacent normal specimens, and their association with clinicopathological factors. We showed that the expression levels of C21orF96 were associated with gross appearance, lymphatic metastasis and distal metastasis. The effect of C21orF96 was assessed by over-expressing the lncRNA. We also found that C21orF96 promoted the tubular formation, migration and invasion. Together, our results suggest that C21orF96 is an oncogenic lncRNA that promotes tumor progression and plays a pivotal role in the development of gastric cancer.

MicroRNAs (miRNAs) have been integrated into tumorigenic programs by regulating genes at post-transcriptional level. Long non-coding RNAs (lncRNAs) are novel targets for miRNAs. Here, we reported that miR-203 down-regulation was closely linked to advanced clinical features and poor overall survival (OS) of patients with hepatocellular carcinoma. We also confirmed that miR-203 and oncogene ADAM9 (a disintegrin and metalloproteinase 9)/oncogenic long non-coding RNA HULC (highly up-regulated in liver cancer) were inversely expressed in hepatocellular carcinoma (HCC) tissues or cell lines. More intriguingly, up-regulation of miR-203 diminished the expression of ADAM9 and HULC in HCC cancer cells. Over-expression of miR-203 could markedly inhibit cell proliferation, invasion and induce cell apoptosis. Furthermore, we identified that miR-203 modulated ADAM9 and HULC in a novel post-transcriptional regulatory mechanism. Over-expression of HULC partly rescued the miR-203-mediated antitumor effects. These results suggested that miR-203 played tumor suppressive roles by downregulating ADAM9 and HULC and indicated its potential application in cancer treatment.

The presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. Recently, inflammaging has been partially attributed to the persistent activation of immune cells thought to arise from latent viral infection, but the contribution of activated autoreactive T cells towards the development of inflammaging remains unclear. To determine how age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics natural thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by cellular infiltration into non-lymphoid tissues and elevated IL-6 and TNFα levels. Autoreactive T cell clones were detected in the periphery of FoxN1-cKO mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell (Treg) generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related Treg accumulation but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging).

It is well known that age-related involution (shrinkage) of the thymus (a central cellular immune organ) results in decreased output of naive T cells [1]. The insufficiency of naive T cells significantly reduces the T cell receptor repertoire diversity, thereby leading to immunosenescence. However, the deleterious effects of thymic involution extend beyond diminished output. It also increases the release of harmful autoreactive T cells which usually results in autoimmune diseases [2], e.g. the autoimmune regulator gene (Aire) mutation-induced autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy. However, the mechanisms and clinical implications of the aging-induced increased release of autoreactive T cells are not clear, especially since not all elderly individuals develop specific autoimmune diseases. Most aged individuals possess low-grade, but above baseline, sustained pro-inflammatory factors, such as interleukin-6 and -1, tumor necrosis factor alpha, and C-reactive protein. This chronic inflammatory state, associated with the aging process termed “inflammaging”, has been implicated in the poor prognosis of virtually every age-related disease. Studies have shown that inflammaging can be attributed to senescent cell-induced “senescence-associated secretory phenotype” [3], and the progressive activation of immune cells, mainly elicited by latent viral infections (“foreign-reaction”) [4]. A recent study has demonstrated that the increased release of autoreactive T cells from the involuted thymus also contributes to the development of inflammaging via an “self/auto-reaction” [5]. Not only has this study revealed a new source of inflammaging, but it has also demonstrated that the age-related increased release of autoreactive T cells is clinically significant, despite the lack of an overt autoimmune disease. The study [5] focuses on age-related thymic involution and thymic function in the context of establishment of immune tolerance, which is mainly accomplished through two mechanisms: thymocyte negative selection and the generation of thymic CD4+FoxP3+ T regulatory cells (Tregs). Firstly, using a FoxN1 conditional knockout (cKO) mouse model of accelerated thymic involution [6] crossed to a Rag2-GFP reporter mouse (to track newly released T cells, termed recent thymic emigrants, RTEs), it was demonstrated that RTEs derived from the involuted thymus adopt an autoreactive phenotype. Generally, RTEs are not in a highly active state because they have not yet encountered their cognate antigens. The increased activation of CD4+ and CD8+ RTEs from the involuted thymus in the absence of infection suggests that these RTEs are autoreactive T cells that are responding to peripheral self-antigens. To confirm this, an IRBP (interstitial retinol-binding protein) immunization model was used to amplify and detect autoreactive T cell clones within a polyclonal T cell repertoire. Mice with an involuted thymus showed a significant expansion of activated IRBP specific T cells, while they were mostly undetectable in littermate controls with a normal thymus. It is well-known that most autoreactive T clones are eliminated through thymocyte negative selection. Therefore, the question arises as to why so many autoreactive T clones are released from the involuted thymus. Negative selection in the involuted thymus was then examined using a triple genetically engineered mouse model. This was done by transplanting a newborn mouse thymic lobe from a RIP-mOVA+ and FoxN1-cKO background into the kidney capsule of young adult OT-II TCR-transgenic mice. RIP-mOVA is a transgene, which expresses chicken ovalbumin as a neo-self-antigen in an Aire-dependent manner in the thymic medulla where negative selection takes place. The OT-II TCR transgenic mice have CD4+ thymocytes that specifically recognize RIP-mOVA. Once these OT-II TCR transgenic thymocytes encounter RIP-mOVA+ medullary thymic epithelial cells (mTECs), they are signaled to undergo exaggerated negative selection. However, when bone marrow progenitor cells from OT-II TCR transgenic hosts were seeded into the grafted RIP-mOVA+/FoxN1-cKO thymus in the kidney capsule, the exaggerated negative selection was not observed. Furthermore, mTECs from FoxN1-cKO mice were found to have diminished expression of Aire, suggesting that impaired clonal deletion likely results from defects related to Aire-dependent promiscuous gene expression of tissue-specific self-antigens. This finding confirmed that negative selection is impaired in the involuted thymus. In a healthy individual, the escape of a few autoreactive T cells from negative selection is not necessarily an issue because thymus-derived Tregs are able to suppress the autoimmune response [7]. The study went on to ask whether thymic involution also disrupts Treg generation [5]. Treg generation and function were then analyzed. The results showed that thymic involution does not impair Treg generation, but may even enhance it. Peripheral Tregs from the involuted thymus were demonstrated to possess adequate suppressive capabilities. It is known that in naturally aged mice Tregs accumulate in the periphery and inflammatory cell infiltration into non-lymphoid tissues is elevated. To test whether these phenomena result from intrinsic defects related to thymic involution or are controlled by extrinsic features of the aged peripheral microenvironment, naturally aged spleen cells were transplanted into young Rag gene knockout mice, which do not have endogenous T and B cells. The age-related Treg accumulation and enhanced survival via diminished expression of the pro-apoptotic Bim gene in the Tregs could be reversed by the young microenvironment, while the inflammatory cell infiltration into non-lymphoid tissues could not. These data imply that changes in peripheral aged Tregs are non-intrinsic, while inflammatory infiltration is driven by intrinsic defects related to perturbed negative selection. Together, these findings demonstrate that age-related thymic involution is involved not only in immunosenescence associated with the insufficient output of naive T cells, but also in the emergence of inflammaging via the increased release of autoreactive T cells. Therefore, therapeutically targeting thymic involution should present a promising strategy for attenuating chronic inflammation, thereby reducing a major risk factor associated with morbidity and mortality in virtually every chronic age-related disease.

FoxN1 expresses in thymic epithelial cell (TEC) and skin epithelium to regulate their development. Prenatal mutation in FoxN1 results in thymic development failure and nude phenotype, while postnatal insufficient FoxN1 expression induces thymic atrophy, resulting in declined T-lymphopoiesis. Although enhancing FoxN1 expression in aged thymus promotes functional rejuvenation, whether FoxN1 over-expression at early age or ectopic expression in the bone marrow is beneficial for lymphopoiesis is unknown. Using our newly generated R26-STOPflox-FoxN1 (gain-of-function) mutant mice, which have an over-expressed FoxN1 after various promoter-driven Cre-mediated deletion of the roadblock STOPflox, we found that K14Cre-mediated prenatal FoxN1 over-expression resulted in a newborn lethal phenotype, displaying abnormal permeability in the skin. Ubiquitous deletion of the STOPflox mediated by uCreERT progressive leak in infant mice influenced TEC development, and T- and B-lymphopoiesis. Although the K5CreERT mediated FoxN1 over-expression in adult mice may be beneficial for postponing thymic aging, early induction of K5CreERT activation in infants induced adverse influences to thymoycte and hair follicle development. Therefore, FoxN1 has a stage and tissue sensitivity. Over- and ectopic-expression of FoxN1 at early life adversely influences immature thymic, T-, and B-cell, and skin epithelial development.

Abstract The thymus maintains immunotolerance to self-antigen by deleting self-reactive T cells and generating nTregs. The thymus involutes with age, which is driven by the loss of FoxN1. Thymic involution is thought to be linked with increased susceptibility to autoimmune disease. However, how the age-related loss of FoxN1 induced thymic involution impacts autoimmunity remains unclear. We used a FoxN1 cKO mouse model that mimics natural thymic involution. We found impaired negative selection in the FoxN1 cKO thymus, evidenced by an increase in the frequency of CD4 and CD8 SP thymocytes and a decrease in Aire+ mTECs. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they may be self-reactive T cells. Additionally, we found that nTreg frequency was increased in the thymus, but not in the spleen. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells into young Rag2-/- mice. We found that the young periphery was able to reverse Treg accumulation. Additionally, the adoptive transfer led to an increase in salivary infiltration independent of peripheral age. We conclude that loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, the age-related accumulation of Tregs depends on the age of the microenvironment in which they stay.

The postnatal thymic epithelial progenitor (TEP) pool is proposed to be regulated by the p63 and FoxN1 genes through proliferation and differentiation, respectively. However, the combined role of these two genes in the aging TEP is still a mystery. Evidence from murine models has elucidated contrasting roles of the p63 isoforms during the aging process. We found that TAp63+, but not ΔNp63+, thymic epithelial cells (TECs) were increased with age, accompanied with increased senescence associated β-gal clusters and p21+ TECs. Senescent clusters also developed after intrathymic infusion of exogenous TAp63 cDNA into young wild-type mice. Using our conditional FoxN1 gene knockout mouse model to disrupt TEP differentiation accelerated this senescent phenotype to early middle age. However, upon infusion of exogenous FoxN1 cDNA into aged wild-type mice resulted in only an increase in ΔNp63+ TECs, but no change in TAp63+ TECs in the partially rejuvenated aged thymus. Interestingly, using a novel FoxN1 transgenic mouse model to enhance TEP differentiation, ΔNp63+ TECs were decreased in young thymus. Additionally, the TAp63+ population contained a high percentage of phosphorylated-p53 and apoptotic TECs, but showed no changes in BrdU-labeled proliferation. As a result, FoxN1 controlled TEC differentiation as a bottleneck to determine TEP pool via affecting TAp63 and ΔNp63 levels. Thus, TEC homeostasis during aging has been determined through the p63-FoxN1 regulatory axis.