Promotion of an Antitumor Immune Program by a Tumor-specific, Complement-activating Antibody.

Tumor-targeting Abs can be used to initiate an antitumor immune program, which appears essential to achieve a long-term durable clinical response to cancer. We previously identified an anti-complement factor H (CFH) autoantibody associated with patients with early-stage non-small cell lung cancer. We cloned from their peripheral B cells an mAb, GT103, that specifically recognizes CFH on tumor cells. Although the underlying mechanisms are not well defined, GT103 targets a conformationally distinct CFH epitope that is created when CFH is associated with tumor cells, kills tumor cells in vitro, and has potent antitumor activity in vivo. In the effort to better understand how an Ab targeting a tumor epitope can promote an effective antitumor immune response, we used the syngeneic CMT167 lung tumor C57BL/6 mouse model, and we found that murinized GT103 (mGT103) activates complement and enhances antitumor immunity through multiple pathways. It creates a favorable tumor microenvironment by decreasing immunosuppressive regulatory T cells and myeloid-derived suppressor cells, enhances Ag-specific effector T cells, and has an additive antitumor effect with anti-PD-L1 mAb. Furthermore, the immune landscape of tumors from early-stage patients expressing the anti-CFH autoantibody is associated with an immunologically active tumor microenvironment. More broadly, our results using an mAb cloned from autoantibody-expressing B cells provides novel, to our knowledge, mechanistic insights into how a tumor-specific, complement-activating Ab can generate an immune program to kill tumor cells and inhibit tumor growth.

Impact of HER2-low status for patients with early-stage breast cancer and non-pCR after neoadjuvant chemotherapy: a National Cancer Database Analysis.

PURPOSE: To investigate potential differences in pathological complete response (pCR) rates and overall survival (OS) between HER2-low and HER2-zero patients with early-stage hormone receptor (HR)-positive and triple-negative breast cancer (TNBC), in the neoadjuvant chemotherapy setting. METHODS: We identified early-stage invasive HER2-negative BC patients who received neoadjuvant chemotherapy diagnosed between 2010 and 2018 in the National Cancer Database. HER2-low was defined by immunohistochemistry (IHC) 1+ or 2+ with negative in situ hybridization, and HER2-zero by IHC0. All the methods were applied separately in the HR-positive and TNBC cohorts. Logistic regression was used to estimate the association of HER2 status with pCR (i.e. ypT0/Tis and ypN0). Kaplan-Meier method and Cox proportional hazards model were applied to estimate the association of HER2 status with OS. Inverse probability weighting and/or multivariable regression were applied to all analyses. RESULTS: For HR-positive patients, 70.9% (n = 17,934) were HER2-low, whereas 51.1% (n = 10,238) of TNBC patients were HER2-low. For both HR-positive and TNBC cohorts, HER2-low status was significantly associated with lower pCR rates [HR-positive: 5.0% vs. 6.7%; weighted odds ratio (OR) = 0.81 (95% CI: 0.72-0.91), p < 0.001; TNBC: 21.6% vs. 24.4%; weighted OR = 0.91 (95% CI: 0.85-0.98), p = 0.007] and improved OS [HR-positive: weighted hazard ratio = 0.85 (95% CI: 0.79-0.91), p < 0.001; TNBC: weighted hazard ratio = 0.91 (95% CI: 0.86-0.96), p < 0.001]. HER2-low status was associated with favorable OS among patients not achieving pCR [HR-positive: adjusted hazard ratio = 0.83 (95% CI: 0.77-0.89), p < 0.001; TNBC: adjusted hazard ratio = 0.88 (95% CI 0.83-0.94), p < 0.001], while no significant difference in OS was observed in patients who achieved pCR [HR-positive: adjusted hazard ratio = 1.00 (95% CI: 0.61-1.63), p > 0.99; TNBC: adjusted hazard ratio = 1.11 (95% CI: 0.85-1.45), p = 0.44]. CONCLUSION: In both early-stage HR-positive and TNBC patients, HER2-low status was associated with lower pCR rates. HER2-zero status might be considered an adverse prognostic factor for OS in patients not achieving pCR.

A Potential Role of Interleukin 10 in COVID-19 Pathogenesis.

A unique feature of the cytokine storm in coronavirus disease 2019 (COVID-19) is the dramatic elevation of interleukin 10 (IL-10). This was thought to be a negative feedback mechanism to suppress inflammation. However, several lines of clinical evidence suggest that dramatic early proinflammatory IL-10 elevation may play a pathological role in COVID-19 severity.

Bcl2l1 Deficiency in Osteoblasts Reduces the Trabecular Bone Due to Enhanced Osteoclastogenesis Likely through Osteoblast Apoptosis.

Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.

Selective autophagy of the adaptor protein Bcl10 modulates T cell receptor activation of NF-κB.

The adaptor protein Bcl10 is a critically important mediator of T cell receptor (TCR)-to-NF-κB signaling. Bcl10 degradation is a poorly understood biological phenomenon suggested to reduce TCR activation of NF-κB. Here we have shown that TCR engagement triggers the degradation of Bcl10 in primary effector T cells but not in naive T cells. TCR engagement promoted K63 polyubiquitination of Bcl10, causing Bcl10 association with the autophagy adaptor p62. Paradoxically, p62 binding was required for both Bcl10 signaling to NF-κB and gradual degradation of Bcl10 by autophagy. Bcl10 autophagy was highly selective, as shown by the fact that it spared Malt1, a direct Bcl10 binding partner. Blockade of Bcl10 autophagy enhanced TCR activation of NF-κB. Together, these data demonstrate that selective autophagy of Bcl10 is a pathway-intrinsic homeostatic mechanism that modulates TCR signaling to NF-κB in effector T cells. This homeostatic process may protect T cells from adverse consequences of unrestrained NF-κB activation, such as cellular senescence.

Tumor-associated antigen-based personalized dendritic cell vaccine in solid tumor patients.

Tumor-associated antigens (TAAs) have been tested in various clinical trials in cancer treatment but the patterns of specific T cell response to personalized TAA immunization remains to be fully understood. We report antigen-specific T cell responses in patients immunized with dendritic cell vaccines pulsed with personalized TAA panels. Tumor samples from patients were first analyzed to identify overexpressed TAAs. Autologous DCs were then transfected with pre-manufactured mRNAs encoding the full-length TAAs, overexpressed in the patients' tumors. Patients with glioblastoma multiforme (GBM) or advanced lung cancer received DC vaccines transfected with personalized TAA panels, in combination with low-dose cyclophosphamide, poly I:C, imiquimod and anti-PD-1 antibody. Antigen-specific T cell responses were measured. Safety and efficacy were evaluated. A total of ten patients were treated with DC vaccines transfected with personalized TAA panels containing 3-13 different TAAs. Among the seven patients tested for anti-TAA T cell responses, most of the TAAs induced antigen-specific CD4+ and/or CD8+ T cell responses, regardless of their expression levels in the tumor tissues. No Grade III/IV adverse events were observed among these patients. Furthermore, the treated patients were associated with favorable overall survival when compared to patients who received standard treatment in the same institution. Personalized TAA immunization-induced-specific CD4+ and CD8+ T cell responses without obvious autoimmune adverse events and was associated with favorable overall survival. These results support further studies on DC immunization with personalized TAA panels for combined immunotherapeutic regimens in solid tumor patients.Trial registration ClinicalTrials.gov, NCT02709616 (March, 2016), NCT02808364 (June 2016), NCT02808416 (June, 2016).

Myeloid-Specific Deletion of Mcl-1 Yields Severely Neutropenic Mice That Survive and Breed in Homozygous Form.

Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2 Cre/Cre Mcl1 flox/flox (Mcl1 ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1 ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1 ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1 ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1 ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1 flox/flox (Mcl1 ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1 ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

Molecular Docking and Molecular Dynamics (MD) Simulation of Human Anti-Complement Factor H (CFH) Antibody Ab42 and CFH Polypeptide.

An understanding of the interaction between the antibody and its targeted antigen and knowing of the epitopes are critical for the development of monoclonal antibody drugs. Complement factor H (CFH) is implied to play a role in tumor growth and metastasis. An autoantibody to CHF is associated with anti-tumor cell activity. The interaction of a human monoclonal antibody Ab42 that was isolated from a cancer patient with CFH polypeptide (pCFH) antigen was analyzed by molecular docking, molecular dynamics (MD) simulation, free energy calculation, and computational alanine scanning (CAS). Experimental alanine scanning (EAS) was then carried out to verify the results of the theoretical calculation. Our results demonstrated that the Ab42 antibody interacts with pCFH by hydrogen bonds through the Tyr315, Ser100, Gly33, and Tyr53 residues on the complementarity-determining regions (CDRs), respectively, with the amino acid residues of Pro441, Ile442, Asp443, Asn444, Ile447, and Thr448 on the pCFH antigen. In conclusion, this study has explored the mechanism of interaction between Ab42 antibody and its targeted antigen by both theoretical and experimental analysis. Our results have important theoretical significance for the design and development of relevant antibody drugs.

Lung inflammation stalls Th17-cell migration en route to the central nervous system during the development of experimental autoimmune encephalomyelitis.

Recruiting pathogenic T cells to the central nervous system (CNS) is a critical step during the development of experimental autoimmune encephalomyelitis (EAE). Here, we report that the absence of autophagy and microtubule-associated protein 1A/1B-light chain 3-associated phagocytosis significantly delayed the onset of EAE in Atg7 conditional knockout (Atg7 CKO) mice in myeloid cells. T-helper cell-cell priming appeared to be normal in the Atg7 CKO mice, but the mice showed significant accumulation of Th17 cells in the lung. The data suggested that the stalling of Th17 cells in the lung en route to the CNS caused the delay. The lung of Atg7 CKO mice, in which we previously demonstrated spontaneous mild inflammation, showed high expression of CCL20, a chemokine that attracts Th17 cells. We have also shown that LPS intranasal instillation delayed EAE onset, suggesting that pulmonary inflammation has an impact on EAE development. Based on our data, therapeutic immunomodulation targeted to the lung, rather than systemically, might be a possible future option to treat multiple sclerosis.

c-FLIP protects T lymphocytes from apoptosis in the intrinsic pathway.

Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway) or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies suggest that cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) promotes cell survival in death receptor-induced apoptosis pathway in T lymphocytes. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of effector caspases. Whether c-FLIP regulates mitochondrion-dependent apoptotic signals remains unknown. In this study, c-FLIP gene was deleted in mature T lymphocytes in vitro, and the role of c-FLIP protein in intrinsic apoptosis pathway was studied. In resting T cells treated with the intrinsic apoptosis inducer, c-FLIP suppressed cytochrome c release from mitochondria. Bim-deletion rescued the enhanced apoptosis in c-FLIP-deficient T cells, whereas inhibition of caspase 8 did not. Different from activated T cells, there was no necroptosis or increase in reactive oxygen species in c-FLIP-deficient resting T cells. These data suggest that c-FLIP is a negative regulator of intrinsic apoptosis pathway in T lymphocytes.

The lung is protected from spontaneous inflammation by autophagy in myeloid cells.

The lung is constantly exposed to the outer environment; thus, it must maintain a state of immune ignorance or tolerance not to overrespond to harmless environmental stimuli. How cells in the lung control immune responses under nonpathogenic condition is not fully understood. In this study, we found that autophagy plays a critical role in the lung-specific immune regulation that prevents spontaneous inflammation. Autophagy in pulmonary myeloid cells plays a role in maintaining low burdens of environmental microbes in the lung, as well as in lowering mitochondrial reactive oxygen species production and preventing overresponse to TLR4 ligands in alveolar macrophages. Based on these mechanisms, we also found that intranasal instillation of antibiotics or an inhibitor of reactive oxygen species was efficient in preventing spontaneous pulmonary inflammation. Thus, autophagy in myeloid cells, particularly alveolar macrophages, is critical for inhibiting spontaneous pulmonary inflammation, and pulmonary inflammation caused by dysfunctional autophagy is pharmacologically prevented.

Cellular FLIP Inhibits Myeloid Cell Activation by Suppressing Selective Innate Signaling.

Cellular FLIP (c-FLIP) specifically inhibits caspase-8 and suppresses death receptor-induced apoptosis. c-FLIP has also been reported to transmit activation signals. In this study, we report a novel function of c-FLIP involving inhibition of myeloid cell activation through antagonizing the selective innate signaling pathway. We found that conditional knockout of c-FLIP in dendritic cells (DCs) led to neutrophilia and splenomegaly. Peripheral DC populations, including CD11b(+) conventional DCs (cDCs), CD8(+) cDCs, and plasmacytoid DCs, were not affected by c-FLIP deficiency. We also found that c-FLIP knockout cDCs, plasmacytoid DCs, and bone marrow-derived DCs (BMDCs) displayed enhanced production of TNF-α, IL-2, or G-CSF in response to stimulation of TLR4, TLR2, and dectin-1. Consistent with the ability of c-FLIP to inhibit the activation of p38 MAPK, the enhanced activation of c-FLIP-deficient BMDCs could be partly linked to an elevated activation of p38 MAPK after engagement of innate receptors. Increased activation was also found in c-FLIP(+/-) macrophages. Additionally, the increased activation in c-FLIP-deficient DCs was independent of caspase-8. Our results reveal a novel inhibitory role of c-FLIP in myeloid cell activation and demonstrate the unexpected anti-inflammatory activity of c-FLIP. Additionally, our observations suggest that cancer therapy targeting c-FLIP downregulation may facilitate DC activation and increase T cell immunity.

The contribution of autophagy to lymphocyte survival and homeostasis.

Over the life span of a T lymphocyte, from thymic development to death, it is subjected to a variety of stresses and stimuli. Upon receipt of each stress or stimulus, a potentially life-changing fate decision must be made, namely, whether to commit to a form of programmed cell death or to make the necessary adaptations to effectively deal with the changing environment. In our laboratory, we have identified several stresses that a T lymphocyte will encounter during a normal life span. Our studies have focused on how T cells utilize autophagy to get a grasp on the situation, or in cases in which survival is untenable, how T cells use autophagy to hasten their demise. This review focuses on the functions of T-cell autophagy in maintaining homeostasis, eliminating excess or dangerous levels of mitochondria, trimming levels of endoplasmic reticulum, and promoting a healthy metabolic level to allow cells to perform as productive components of the immune system. In addition, the use of autophagy signaling molecules to perform autophagy-independent tasks involved in the maintenance of immune homeostasis is discussed.

The c-FLIPL cleavage product p43FLIP promotes activation of extracellular signal-regulated kinase (ERK), nuclear factor κB (NF-κB), and caspase-8 and T cell survival.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.

Plasma microRNA signature as a noninvasive biomarker for acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is the leading cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Approximately 35% to 50% of HCT recipients develop aGVHD; however, there are no validated diagnostic and predictive blood biomarkers for aGVHD in clinical use. Here, we show that plasma samples from aGVHD patients have a distinct microRNA (miRNA) expression profile. We found that 6 miRNAs (miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a) were significantly upregulated in the plasma of aGVHD patients (n = 116) when compared with non-GVHD patients (n = 52) in training and validation phases. We have developed a model including 4 miRNAs (miR-423, miR-199a-3p, miR-93*, and miR-377) that can predict the probability of aGVHD with an area under the curve of 0.80. Moreover, these elevated miRNAs were detected before the onset of aGVHD (median = 16 days before diagnosis). In addition, the levels of these miRNAs were positively associated with aGVHD severity, and high expression of the miRNA panel was associated with poor overall survival. Furthermore, the miRNA signature for aGVHD was not detected in the plasma of lung transplant or nontransplant sepsis patients. Our results have identified a specific plasma miRNA signature that may serve as an independent biomarker for the prediction, diagnosis, and prognosis of aGVHD.

Transfer of CD8+ T cell memory using Bcl-2 as a marker.

The processes that regulate T cell memory generation are important for therapeutic design and the immune response to disease. However, what allows a subset of effector T cells to survive the contraction period to become memory cells is incompletely understood. The Bcl-2 family is critical for T cell survival, and Bcl-2 has been proposed to be important for the survival of memory cells. However, previous studies have relied on double-knockout models, potentially skewing the role of Bcl-2, and the use of Bcl-2 as a marker in adoptive transfer experiments, a method required to confirm the memory potential of cell subsets, has not been possible because of the intracellular localization of the protein. In this study, we present a novel Bcl-2 reporter mouse model and, to our knowledge, show for the first time that a distinct subset of effector T cells, and also a subset within the CD127(hi)KLRG1(lo) memory precursor effector cell population, retains high Bcl-2 expression at the peak of the CD8(+) T cell response to Listeria monocytogenes. Furthermore, we show that Bcl-2 correlates with memory potential in adoptive transfer experiments using both total responding CD8(+) T cells and memory precursor effector cells. These results show that even within the memory precursor effector cell population, Bcl-2 confers a survival advantage in a subset of effector CD8(+) T cells that allows differentiation into memory cells and cement Bcl-2 as a critical factor for T cell memory.

Autophagic activity dictates the cellular response to oncogenic RAS.

RAS is frequently mutated in human cancers and has opposing effects on autophagy and tumorigenesis. Identifying determinants of the cellular responses to RAS is therefore vital in cancer research. Here, we show that autophagic activity dictates the cellular response to oncogenic RAS. N-terminal Apoptosis-stimulating of p53 protein 2 (ASPP2) mediates RAS-induced senescence and inhibits autophagy. Oncogenic RAS-expressing ASPP2((Δ3/Δ3)) mouse embryonic fibroblasts that escape senescence express a high level of ATG5/ATG12. Consistent with the notion that autophagy levels control the cellular response to oncogenic RAS, overexpressing ATG5, but not autophagy-deficient ATG5 mutant K130R, bypasses RAS-induced senescence, whereas ATG5 or ATG3 deficiency predisposes to it. Mechanistically, ASPP2 inhibits RAS-induced autophagy by competing with ATG16 to bind ATG5/ATG12 and preventing ATG16/ATG5/ATG12 formation. Hence, ASPP2 modulates oncogenic RAS-induced autophagic activity to dictate the cellular response to RAS: to proliferate or senesce.

Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8.

BACKGROUND & AIMS: The intestinal epithelium generates a barrier that protects mammals from potentially harmful intestinal contents, such as pathogenic bacteria. Dysregulation of epithelial cell death has been implicated in barrier dysfunction and in the pathogenesis of intestinal inflammation. We investigated mechanisms of cell-death regulation in the intestinal epithelium of mice. METHODS: Conditional knockout mice (either inducible or permanent) with deletion of cellular FLICE-inhibitory protein (cFlip) or caspase-8 in the intestinal epithelium were analyzed by histology and high-resolution endoscopy. We assessed the effects of cFlip or caspase-8 deficiency on intestinal homeostasis. RESULTS: Expression of cFlip in the intestinal epithelium was required for constitutive activation of caspase-8 under steady-state conditions. Intestinal expression of cFlip was required for development; disruption of the gene encoding cFlip from the intestinal epithelium (cFlip(fl/fl) VillinCre(+) mice) resulted in embryonic lethality. When cFlip was deleted from the intestinal epithelium of adult mice (cFlip(iΔIEC) mice), the animals died within a few days from severe tissue destruction, epithelial cell death, and intestinal inflammation. Death of cFlip-depleted intestinal epithelial cells was regulated extrinsically and required the presence of death receptor ligands, such as tumor necrosis factor-α and CD95 ligand, but was independent of receptor-interacting protein 3. cFlip deficiency was associated with strong up-regulation of caspase-8 and caspase-3 activity and excessive apoptosis in intestinal crypts. CONCLUSIONS: cFlip is required for intestinal tissue homeostasis in mice. It controls the level of activation of caspase-8 to promote survival of intestinal epithelial cells.