Hongni Fan

Developing a pro-regenerative biomaterial scaffold microenvironment requires T helper 2 cells

Engineering a healing immune response Infections, surgeries, and trauma can all cause major tissue damage. Biomaterial scaffolds, which help to guide regenerating tissue, are an exciting emerging therapeutic strategy to promote tissue repair. Sadtler et al. tested how biomaterial scaffolds interact with the immune system in damaged tissue to promote repair (see the Perspective by Badylak). Scaffolds derived from cardiac muscle and bone extracellular matrix components trigger a tissue-reparative T cell immune response in mice with injured muscles. Science, this issue p. 366; see also p. 298 Biomaterial scaffolds engage the immune system to promote tissue repair. [Also see Perspective by Badylak] Immune-mediated tissue regeneration driven by a biomaterial scaffold is emerging as an innovative regenerative strategy to repair damaged tissues. We investigated how biomaterial scaffolds shape the immune microenvironment in traumatic muscle wounds to improve tissue regeneration. The scaffolds induced a pro-regenerative response, characterized by an mTOR/Rictor-dependent T helper 2 pathway that guides interleukin-4–dependent macrophage polarization, which is critical for functional muscle recovery. Manipulating the adaptive immune system using biomaterials engineering may support the development of therapies that promote both systemic and local pro-regenerative immune responses, ultimately stimulating tissue repair.

Regulatory T-cell Response to Enterotoxigenic Bacteroides fragilis Colonization Triggers IL17-Dependent Colon Carcinogenesis

UNLABELLED Many epithelial cancers are associated with chronic inflammation. However, the features of inflammation that are procarcinogenic are not fully understood. Regulatory T cells (Treg) typically restrain overt inflammatory responses and maintain intestinal immune homeostasis. Their immune-suppressive activity can inhibit inflammation-associated cancers. Paradoxically, we show that colonic Tregs initiate IL17-mediated carcinogenesis in multiple intestinal neoplasia mice colonized with the human symbiote enterotoxigenic Bacteroides fragilis (ETBF). Depletion of Tregs in ETBF-colonized C57BL/6 FOXP3(DTR) mice enhanced colitis but diminished tumorigenesis associated with shifting of mucosal cytokine profile from IL17 to IFNγ; inhibition of ETBF-induced colon tumorigenesis was dependent on reduced IL17 inflammation and was independent of IFNγ. Treg enhancement of IL17 production is cell-extrinsic. IL2 blockade restored Th17 responses and tumor formation in Treg-depleted animals. Our findings demonstrate that Tregs limit the availability of IL2 in the local microenvironment, allowing the Th17 development necessary to promote ETBF-triggered neoplasia, and thus unveil a new mechanism whereby Treg responses to intestinal bacterial infection can promote tumorigenesis. SIGNIFICANCE Tregs promote an oncogenic immune response to a common human symbiote associated with inflammatory bowel disease and colorectal cancer. Our data define mechanisms by which mucosal Tregs, despite suppressing excessive inflammation, promote the earliest stages of immune procarcinogenesis via enhancement of IL17 production at the expense of IFNγ production.

Redundant Innate and Adaptive Sources of IL17 Production Drive Colon Tumorigenesis

IL17-producing Th17 cells, generated through a STAT3-dependent mechanism, have been shown to promote carcinogenesis in many systems, including microbe-driven colon cancer. Additional sources of IL17, such as γδ T cells, become available under inflammatory conditions, but their contributions to cancer development are unclear. In this study, we modeled Th17-driven colon tumorigenesis by colonizing Min(Ap) (c+/-) mice with the human gut bacterium, enterotoxigenic Bacteroides fragilis (ETBF), to investigate the link between inflammation and colorectal cancer. We found that ablating Th17 cells by knocking out Stat3 in CD4(+) T cells delayed tumorigenesis, but failed to suppress the eventual formation of colonic tumors. However, IL17 blockade significantly attenuated tumor formation, indicating a critical requirement for IL17 in tumorigenesis, but from a source other than Th17 cells. Notably, genetic ablation of γδ T cells in ETBF-colonized Th17-deficient Min mice prevented the late emergence of colonic tumors. Taken together, these findings support a redundant role for adaptive Th17 cell- and innate γδT17 cell-derived IL17 in bacteria-induced colon carcinogenesis, stressing the importance of therapeutically targeting the cytokine itself rather than its cellular sources. Cancer Res; 76(8); 2115-24. ©2016 AACR.

IFN-producing killer dendritic cells are antigen-presenting cells endowed with T-cell cross-priming capacity

IFN-producing killer dendritic cells (IKDC) represent a recently discovered cell type in the immune system that possesses a number of functions contributing to innate and adaptive immunity, including production of type 1 and 2 IFNs, interleukin (IL)-12, natural killing, and ultimately antigen presentation to naïve T cells. Here, we compared in vitro and in vivo responses of mouse IKDC, conventional dendritic cells (DC), and natural killer (NK) cells to murine cytomegalovirus infection and found distinct functions among these cell subsets. Upon recognition of infected fibroblasts, IKDC, as well as NK, produced high level of IFN-gamma, but unlike NK, IKDC simultaneously produced IL-12p40 and up-regulated MHC class II (MHC-II) and costimulatory molecules. Using MHC-II molecule expression as a phenotypic marker to distinguish activated IKDC from activated NK, we further showed that highly purified MHC-II(+) IKDC but not NK cross-present MHC class I-restricted antigens derived from MCMV-infected targets to CD8(+) T cells in vitro and in vivo. Our findings emphasize the unique nature of IKDC as a killer antigen-presenting cell directly linking innate and adaptive immunity.

The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis.

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of interleukin-17 (IL-17)-dependent colon tumorigenesis in MinApc+/− mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell-sorted immature myeloid cells were functionally assayed for inhibition of T-cell proliferation and inducible nitric oxide synthase expression to delineate MDSC populations. A comparison of ETBF infection with that of other oncogenic bacteria (Fusobacterium nucleatum or pks+Escherichia coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic-MDSCs (MO-MDSCs). Combined action of the B. fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO, and suppressed T-cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.

Targeted inactivation of copper transporter Atp7b in hepatocytes causes liver steatosis and obesity in mice

Copper-transporting ATPase 2 (ATP7B) is essential for mammalian copper homeostasis. Mutations in ATP7B result in copper accumulation, especially in the liver, and cause Wilson disease (WD). The major role of hepatocytes in WD pathology is firmly established. It is less certain whether the excess Cu in hepatocytes is solely responsible for development of WD. To address this issue, we generated a mouse strain for Cre-mediated deletion of Atp7b and inactivated Atp7b selectively in hepatocytes. Atp7bΔHep mice accumulate copper in the liver, have elevated urinary copper, and lack holoceruloplasmin but show no liver disease for up to 30 wk. Liver inflammation is muted and markedly delayed compared with the age-matched Atp7b-/- null mice, which show a strong type1 inflammatory response. Expression of metallothioneins is higher in Atp7bΔHep livers than in Atp7b-/- mice, suggesting better sequestration of excess copper. Characterization of purified cell populations also revealed that nonparenchymal cells in Atp7bΔHep liver maintain Atp7b expression, have normal copper balance, and remain largely quiescent. The lack of inflammation unmasked metabolic consequences of copper misbalance in hepatocytes. Atp7bΔHep animals weigh more than controls and have higher levels of liver triglycerides and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase. By 45 wk, all animals develop liver steatosis on a regular diet. Thus copper misbalance in hepatocytes dysregulates lipid metabolism, whereas development of inflammatory response in WD may depend on copper status of nonparenchymal cells. The implications of these findings for the cell-targeting WD therapies are discussed.NEW & NOTEWORTHY Targeted inactivation of copper-transporting ATPase 2 (Atp7b) in hepatocytes causes steatosis in the absence of inflammation.

Non-toxigenic Bacteroides fragilis (NTBF) administration reduces bacteria-driven chronic colitis and tumor development independent of polysaccharide A

Polysaccharide A (PSA), an immunogenic capsular component of non-toxigenic Bacteroides fragilis (NTBF) strain NCTC 9343, is reported to promote mucosal immune development and suppress colitis. Contrastingly, enterotoxigenic Bacteroides fragilis (ETBF) is highly associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC), rapidly inducing IL-17-dependent murine colitis and tumorigenesis. In specific-pathogen-free (SPF) C57BL/6 wild-type (WT) and multiple intestinal neoplasia (MinApc716+/−) mice, we show that sequential treatment of the NTBF strain, 9343, followed by the ETBF strain, 86-5443-2-2 (86), diminished colitis and tumorigenesis. Mice treated simultaneously with 9343 and 86 exhibited both severe colitis and tumorigenesis. Abrogated disease severity in sequentially treated mice was attributed to 9343 strain dominance and decreased IL-17A, but 86 colonization prior to or simultaneous with 9343 mitigated the anti-inflammatory effect of 9343. Remarkably, 9343-mediated protection was independent of PSA, as sequentially treated mice receiving ΔPSA 9343 exhibited similar protection. Further, SPF WT and Min mice colonized with PSA-competent or PSA-deficient 9343 exhibited similar IL-10, IL-17, and IFN-γ responses. Treatment of 86-colonized mice with 9343 failed to disrupt 86 pathogenesis. Our findings demonstrate that 9343 colonization, independent of PSA, offers prophylaxis against colitis-inducing 86 but may not be a valid therapy once colitis is established.

Abstract A141: The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-gamma production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Our findings are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Furthermore, our results were clinically validated in a phase 2 study at Hopkins which showed mismatch-repair status as a predictor of clinical benefit to immune checkpoint blockade with pembrolizumab. Citation Format: Nicolas J. Llosa, Franck Housseau, Elizabeth Wick, Hechenbleikner Lizzy, Michael Cruise, Robert Anders, Cynthia Sears, Drew M. Pardoll, Hongni Fan, Nicholas Siegel, Lee Blosser, Ada Tam, Hao Wang. The vigorous immune microenvironment of microsatellite instable colon cancer isbalanced by multiple counter-inhibitory checkpoints. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A141.

Abstract A088: Immune profiling of inflamed microsatellite stable colorectal cancer

Immunotherapy is an important therapeutic modality that is rapidly turning into standard of care for many cancers. However, early immunotherapy efforts in colorectal cancer (CRC) were ineffective, and only recently did a successful breakthrough lead to improved survival in a small subset of metastatic colorectal cancer patients which were noted to harbor MMR deficiency (microsatellite instability: MSI). Therefore, there is an urgent need for additional biomarkers to identify CRC patients who are predicted to respond to immunotherapy. For the past year we have been interrogating the immune microenvironment of microsatellite stable (MSS) CRC patients to aid our understanding of why a fair proportion of these patients have an inflamed tumor microenvironment (TME). We have banked 140 tumor/normal CRC tissue matched pairs using our recruitment network at Hopkins. Genetic MSI testing was done on 114 of these patients, with 20 (17%) and 94 (83%) being categorized as MSI and MSS, respectively. Our results are therefore representative of the general CRC population which includes 15-20% of MSI + tumors. We are also currently working with material obtained from CRC patients treated under the Hopkins phase 2 study testing anti PD-1 therapy in patients with MSI tumors. Immunohistochemistry (IHC) and digital image analysis of primary and trial CRC patients: We previously demonstrated the critical geographic association between infiltrating CD8 + T cells and immuno-regulatory molecule expression in the TME of MSI CRC. CRC FFPE tissue sections were stained with anti-CD3 and anti-CD8 antibodies and digital image analysis utilizing the HALO platform was performed. Inflamed MSS CRC are infiltrated by PD1 high T cells which are functionally suppressed: Multiparameter flow cytometry (MFC) performed on lymphocytes freshly isolated from our primary CRC tumors demonstrated that a large proportion of both CD4 + and CD8 + T cells infiltrating what we denominate inflamed MSS tumors express higher levels of PD-1 compared to conventional MSS patients. Very importantly, these PD-1 high T cells are capable of producing a large amount of IFN-γ after short term PMA/ionomycin stimulation. CD8 + T cells co-localize with immune checkpoint expression in inflamed MSS CRC: We further explored the nature of the T-cell infiltrates by performing laser capture microdissection (LCM) on inflamed MSS tumors and MSS patients who stabilized disease during anti PD-1 therapy, separately dissecting the TIL and invasive front compartments and then performed qRT-PCR for selected genes encoding signature T-cell cytokines as well as core transcription factors for each of the three major Th subsets, Th1/Tc1 (type I CTL; TBX21 and IFN-γ are common to Th1 and Tc1), Th2, and Th17. We additionally analyzed genes associated with CTL and Treg and also general inflammatory cytokines. Finally, we analyzed expression of genes encoding both co-inhibitory membrane ligands and receptors (checkpoints) and metabolic enzymes that have been shown to regulate lymphocyte activity. Inflamed MSS tumors exhibited a similar CD8 + T cell infiltration with a Th1/Tc1 immune signature associated with the counter expression of immune checkpoints as observed in MSI patients. Prediction of MHC class I-restricted Mutation Associated Neoantigens (MANAs): Nonsynonymous mutations in tumor tissue are expected to generate MANAs, which are 8-11 amino-acid peptides generated from proteosomal degradation and recognized by tumor-specific CD8 + T cells in the context of HLA-I restriction. By comparing tumor to normal colon exome we are currently identifying mutations and MANAs in MSS CRC patients who showed high levels of lymphocyte infiltration and PD-1 expression. MANAs in inflamed MSS CRC will be tested for their recognition by autologous TIL. Analysis of the TCR V β repertoire in primary CRC and anti-PD-1-treated CRC: In addition to the IHC characterization of CRC T cell infiltration, we are using TCRVβ clonality analysis of the intra-tumor immune response as a metric of the intensity of the antitumor immune response. We postulate that the number and the frequency of unique TCRVβ sequences in the tumor tissue should reflect the density and the immunogeni city of neoepitopes, respectively. In conclusion, we have identified a subset of MSS CRCs that exhibit an “MSI-like” immunologic microenvironment. The final aim of our studies is to validate that MSS CRC patients with a preexisting anti-tumor immune response can benefit from immunotherapeutic interventions. Citation Format: Nicolas Jose Llosa, Franck Housseau, Nicholas Siegel, Kellie N. Smith, Hongni Fan, Robert M. Anders, Dung Le, Luis Diaz, Jr., Cynthia Sears, Drew M. Pardoll. Immune profiling of inflamed microsatellite stable colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A088.

The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints.

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-gamma production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five - PD-1, PD-L1, CTLA-4, LAG-3 and IDO - currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. Our findings are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair defective subset of CRC selectively up-regulates at least 5 checkpoint molecules that are targets of inhibitors currently being clinically tested. Furthermore, our results were clinically validated in a Phase II study at Hopkins which showed mismatch-repair status as a predictor of clinical benefit to immune checkpoint blockade with pembrolizumab.