SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Introduction: Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). Results: In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Discussion: Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

SMAD4 TGF-ß-independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation.

SMAD4, a mediator of TGF-ß signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-ß receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-ß signature in naive CD8+ T cells. Mechanistically, prior to TGF-ß signaling, SMAD4 binds to promoters and enhancers of several TGF-ß target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-ß signal, SMAD4 limits the expression of TGF-ß negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-ß. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-ß control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-ß-independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.

Antitumor efficacy of combined CTLA4/PD-1 blockade without intestinal inflammation is achieved by elimination of FcγR interactions.

BACKGROUND: Programmed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events. METHODS: We have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody. RESULTS: Sustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit. CONCLUSION: These data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade.

IL-23 and IL-2 activation of STAT5 is required for optimal IL-22 production in ILC3s during colitis.

Signal transducer and activator of transcription (STAT) proteins have critical roles in the development and function of immune cells. STAT signaling is often dysregulated in patients with inflammatory bowel disease (IBD), suggesting the importance of STAT regulation during the disease process. Moreover, genetic alterations in STAT3 and STAT5 (e.g., deletions, mutations, and single-nucleotide polymorphisms) are associated with an increased risk for IBD. In this study, we elucidated the precise roles of STAT5 signaling in group 3 innate lymphoid cells (ILC3s), a key subset of immune cells involved in the maintenance of gut barrier integrity. We show that mice lacking either STAT5a or STAT5b are more susceptible to Citrobacter rodentium-mediated colitis and that interleukin-2 (IL-2)- and IL-23-induced STAT5 drives IL-22 production in both mouse and human colonic lamina propria ILC3s. Mechanistically, IL-23 induces a STAT3-STAT5 complex that binds IL-22 promoter DNA elements in ILC3s. Our data suggest that STAT5a/b signaling in ILC3s maintains gut epithelial integrity during pathogen-induced intestinal disease.

Induction and Analysis of Anti-CD40-induced Colitis in Mice.

Colon inflammation or colitis affects more than 1 million people worldwide. Several pre-clinical models, including chemical-induced (i.e., DSS, TNBS) or pathogen-induced (i.e., Citrobacter rodentium) have been used to study mechanisms involved in the development and regulation of colitis. Anti-CD40 induced colitis model has gained acceptance to study the roles of innate immune cells during acute intestinal inflammation. Here we describe a rapid, robust and reproducible protocol to induce and analyze anti-CD40 mediated colitis in mice.

MAVS deficiency induces gut dysbiotic microbiota conferring a proallergic phenotype.

Prominent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayed-type hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH by modulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs-/- mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies.

LAG3+ Regulatory T Cells Restrain Interleukin-23-Producing CX3CR1+ Gut-Resident Macrophages during Group 3 Innate Lymphoid Cell-Driven Colitis.

Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintains gut homeostasis but can also promote inflammatory bowel disease (IBD). The regulation of ILC3-dependent colitis remains to be elucidated. Here we show that Foxp3+ regulatory T cells (Treg cells) prevented ILC3-mediated colitis in an IL-10-independent manner. Treg cells inhibited IL-23 and IL-1ß production from intestinal-resident CX3CR1+ macrophages but not CD103+ dendritic cells. Moreover, Treg cells restrained ILC3 production of IL-22 through suppression of CX3CR1+ macrophage production of IL-23 and IL-1ß. This suppression was contact dependent and was mediated by latent activation gene-3 (LAG-3)-an immune checkpoint receptor-expressed on Treg cells. Engagement of LAG-3 on MHC class II drove profound immunosuppression of CX3CR1+ tissue-resident macrophages. Our study reveals that the health of the intestinal mucosa is maintained by an axis driven by Treg cells communication with resident macrophages that withhold inflammatory stimuli required for ILC3 function.

Autocrine Adenosine Regulates Tumor Polyfunctional CD73+CD4+ Effector T Cells Devoid of Immune Checkpoints.

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4+ T cells (Teffs) enriched in polyfunctional Th1.17 cells characterized by expression of CXCR3, CCR6, and MDR1, and production of IL17A/IFNγ/IL22/GM-CSF. CD39+ Tregs selectively targeted CD73+ Teffs through cooperative degradation of ATP into Ado inhibiting and restricting the ability of CD73+ Teffs to secrete IL17A. CD73+ Teffs infiltrating breast and ovarian tumors were functionally blunted by Tregs expressing upregulated levels of CD39 and ATPase activity. Moreover, tumor-infiltrating CD73+ Teffs failed to express inhibitory immune checkpoints, suggesting that CD73 might be selected under pressure from immune checkpoint blockade therapy and thus may represent a nonredundant target for restoring antitumor immunity.Significance: Polyfunctional CD73+ T-cell effectors lacking other immune checkpoints are selectively targeted by CD39 overexpressing Tregs that dominate the breast tumor environment. Cancer Res; 78(13); 3604-18. ©2018 AACR.

Transforming growth factor ß: a master regulator of the gut microbiota and immune cell interactions.

The relationship between host organisms and their microbiota has co-evolved towards an inter-dependent network of mutualistic interactions. This interplay is particularly well studied in the gastrointestinal tract, where microbiota and host immune cells can modulate each other directly, as well as indirectly, through the production and release of chemical molecules and signals. In this review, we define the functional impact of transforming growth factor-beta (TGF-ß) on this complex interplay, especially through its modulation of the activity of local regulatory T cells (Tregs), type 17 helper (Th17) cells, innate lymphoid cells (ILCs) and B cells.

Integrin αvß8-Mediated TGF-ß Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation.

Regulatory T (Treg) cells play a pivotal role in suppressing self-harmful T cell responses, but how Treg cells mediate suppression to maintain immune homeostasis and limit responses during inflammation is unclear. Here we show that effector Treg cells express high amounts of the integrin αvß8, which enables them to activate latent transforming growth factor-ß (TGF-ß). Treg-cell-specific deletion of integrin αvß8 did not result in a spontaneous inflammatory phenotype, suggesting that this pathway is not important in Treg-cell-mediated maintenance of immune homeostasis. However, Treg cells lacking expression of integrin αvß8 were unable to suppress pathogenic T cell responses during active inflammation. Thus, our results identify a mechanism by which Treg cells suppress exuberant immune responses, highlighting a key role for effector Treg-cell-mediated activation of latent TGF-ß in suppression of self-harmful T cell responses during active inflammation.

Diversity and expression of microRNAs in the filarial parasite, Brugia malayi.

Human filarial parasites infect an estimated 120 million people in 80 countries worldwide causing blindness and the gross disfigurement of limbs and genitals. An understanding of RNA-mediated regulatory pathways in these parasites may open new avenues for treatment. Toward this goal, small RNAs from Brugia malayi adult females, males and microfilariae were cloned for deep-sequencing. From ∼ 30 million sequencing reads, 145 miRNAs were identified in the B. malayi genome. Some microRNAs were validated using the p19 RNA binding protein and qPCR. B. malayi miRNAs segregate into 99 families each defined by a unique seed sequence. Sixty-one of the miRNA families are highly conserved with homologues in arthropods, vertebrates and helminths. Of those miRNAs not highly conserved, homologues of 20 B. malayi miRNA families were found in vertebrates. Nine B. malayi miRNA families appear to be filarial-specific as orthologues were not found in other organisms. The miR-2 family is the largest in B. malayi with 11 members. Analysis of the sequences shows that six members result from a recent expansion of the family. Library comparisons found that 1/3 of the B. malayi miRNAs are differentially expressed. For example, miR-71 is 5-7X more highly expressed in microfilariae than adults. Studies suggest that in C.elegans, miR-71 may enhance longevity by targeting the DAF-2 pathway. Characterization of B. malayi miRNAs and their targets will enhance our understanding of their regulatory pathways in filariads and aid in the search for novel therapeutics.