Showing papers in "Journal of Experimental Medicine in 2013"

Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.

Abstract: Treatment with monoclonal antibody specific for cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma. Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression. We demonstrate, however, that the activity of anti–CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions. Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor–expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4–based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.

Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF

Abstract: Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac–derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80hiCD11blo primitive macrophages and Ly6ChiCD11bhi fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF–deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.

A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis.

Abstract: Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.

Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages

Abstract: Eosinophils in visceral adipose tissue (VAT) have been implicated in metabolic homeostasis and the maintenance of alternatively activated macrophages (AAMs). The absence of eosinophils can lead to adiposity and systemic insulin resistance in experimental animals, but what maintains eosinophils in adipose tissue is unknown. We show that interleukin-5 (IL-5) deficiency profoundly impairs VAT eosinophil accumulation and results in increased adiposity and insulin resistance when animals are placed on a high-fat diet. Innate lymphoid type 2 cells (ILC2s) are resident in VAT and are the major source of IL-5 and IL-13, which promote the accumulation of eosinophils and AAM. Deletion of ILC2s causes significant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associated with increased adipose ILC2 cytokine production and enhanced insulin sensitivity. Further, IL-33, a cytokine previously shown to promote cytokine production by ILC2s, leads to rapid ILC2-dependent increases in VAT eosinophils and AAMs. Thus, ILC2s are resident in VAT and promote eosinophils and AAM implicated in metabolic homeostasis, and this axis is enhanced during Th2-associated immune stimulation.

Diverse novel functions of neutrophils in immunity, inflammation, and beyond

Abstract: Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10–20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease.

Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production

Abstract: In the present study, we have investigated the distribution of HIV-specific and HIV-infected CD4 T cells within different populations of memory CD4 T cells isolated from lymph nodes of viremic HIV-infected subjects. Four memory CD4 T cell populations were identified on the basis of the expression of CXCR5, PD-1, and Bcl-6: CXCR5(-)PD-1(-)Bcl-6(-), CXCR5(+)PD-1(-)Bcl-6(-), CXCR5(-)PD-1(+)Bcl-6(-), and CXCR5(+)PD-1(+)Bcl-6(+). On the basis of Bcl-6 expression and functional properties (IL-21 production and B cell help), the CXCR5(+)PD-1(+)Bcl-6(+) cell population was considered to correspond to the T follicular helper (Tfh) cell population. We show that Tfh and CXCR5(-)PD-1(+) cell populations are enriched in HIV-specific CD4 T cells, and these populations are significantly increased in viremic HIV-infected subjects as compared with healthy subjects. The Tfh cell population contained the highest percentage of CD4 T cells harboring HIV DNA and was the most efficient in supporting productive infection in vitro. Replication competent HIV was also readily isolated from Tfh cells in subjects with nonprogressive infection and low viremia (<1,000 HIV RNA copies). However, only the percentage of Tfh cells correlated with the levels of plasma viremia. These results demonstrate that Tfh cells serve as the major CD4 T cell compartment for HIV infection, replication, and production.

Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4

Abstract: The cytotoxic T lymphocyte antigen-4 (CTLA-4)–blocking antibody ipilimumab results in durable responses in metastatic melanoma, though therapeutic benefit has been limited to a fraction of patients. This calls for identification of resistance mechanisms and development of combinatorial strategies. Here, we examine the inhibitory role of indoleamine 2,3-dioxygenase (IDO) on the antitumor efficacy of CTLA-4 blockade. In IDO knockout mice treated with anti–CTLA-4 antibody, we demonstrate a striking delay in B16 melanoma tumor growth and increased overall survival when compared with wild-type mice. This was also observed with antibodies targeting PD-1–PD-L1 and GITR. To highlight the therapeutic relevance of these findings, we show that CTLA-4 blockade strongly synergizes with IDO inhibitors to mediate rejection of both IDO-expressing and nonexpressing poorly immunogenic tumors, emphasizing the importance of the inhibitory role of both tumor- and host-derived IDO. This effect was T cell dependent, leading to enhanced infiltration of tumor-specific effector T cells and a marked increase in the effector-to-regulatory T cell ratios in the tumors. Overall, these data demonstrate the immunosuppressive role of IDO in the context of immunotherapies targeting immune checkpoints and provide a strong incentive to clinically explore combination therapies using IDO inhibitors irrespective of IDO expression by the tumor cells.

Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells

Abstract: Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2–TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)–related class I–like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-d-ribityllumazine (rRL-6-CH2OH), specifically detect all human MAIT cells. Tetramer+ MAIT subsets were predominantly CD8+ or CD4−CD8−, although a small subset of CD4+ MAIT cells was also detected. Notably, most human CD8+ MAIT cells were CD8α+CD8β−/lo, implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a TH1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1–rRL-6-CH2OH tetramers detected CD4+, CD4−CD8− and CD8+ MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-β repertoire, and although the majority of human MAIT cells expressed TRAV1-2–TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.

Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model

Abstract: Patients with inflammatory bowel disease (IBD) have an increased risk of colon cancer. However, the immune cells and cytokines that mediate the transition from intestinal inflammation to cancer are poorly understood. We show that bacteria-induced colon cancer is accompanied by differential accumulation of IL-17+IL-22+ colonic innate lymphoid cells (cILCs), which are phenotypically distinct from LTi and NK-22 cells, and that their depletion in mice with dysplastic inflammation blocks the development of invasive colon cancer. Analysis of the functional role of distinct Type 17 cytokines shows that although blockade of IL-17 inhibits some parameters of intestinal inflammation, reduction in dysplasia and colorectal cancer (CRC) requires neutralization of IL-22 indicating a unique role for IL-22 in the maintenance of cancer in this model. Mechanistic analyses showed that IL-22 selectively acts on epithelial cells to induce Stat3 phosphorylation and proliferation. Importantly, we could detect IL-22+CD3+ and IL-22+CD3− cells in human CRC. Our results describe a new activity of IL-22 in the colon as a nonredundant mediator of the inflammatory cascade required for perpetuation of CRC, highlighting the IL-22 axis as a novel therapeutic target in colon cancer.

PDGFRα and CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion

Abstract: The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin+ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45− Ter119− CD31− mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin+ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα+ CD51+ subset of Nestin+ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα+ CD51+ cells in the human fetal BM represent a small subset of CD146+ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα+ CD51+ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.

Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Abstract: Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis

Abstract: Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)- DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLADRB1* 04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLADRB1* 04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1

Abstract: Macrophages (MΦs) colonize tissues during inflammation in two distinct ways: recruitment of monocyte precursors and proliferation of resident cells. We recently revealed a major role for IL-4 in the proliferative expansion of resident MΦs during a Th2-biased tissue nematode infection. We now show that proliferation of MΦs during intestinal as well as tissue nematode infection is restricted to sites of IL-4 production and requires MΦ-intrinsic IL-4R signaling. However, both IL-4Rα–dependent and –independent mechanisms contributed to MΦ proliferation during nematode infections. IL-4R–independent proliferation was controlled by a rise in local CSF-1 levels, but IL-4Rα expression conferred a competitive advantage with higher and more sustained proliferation and increased accumulation of IL-4Rα+ compared with IL-4Rα− cells. Mechanistically, this occurred by conversion of IL-4Rα+ MΦs from a CSF-1–dependent to –independent program of proliferation. Thus, IL-4 increases the relative density of tissue MΦs by overcoming the constraints mediated by the availability of CSF-1. Finally, although both elevated CSF1R and IL-4Rα signaling triggered proliferation above homeostatic levels, only CSF-1 led to the recruitment of monocytes and neutrophils. Thus, the IL-4 pathway of proliferation may have developed as an alternative to CSF-1 to increase resident MΦ numbers without coincident monocyte recruitment.

IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

Abstract: IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)-deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.

p53-dependent chemokine production by senescent tumor cells supports NKG2D-dependent tumor elimination by natural killer cells

Abstract: The induction of cellular senescence is an important mechanism by which p53 suppresses tumorigenesis. Using a mouse model of liver carcinoma, where cellular senescence is triggered in vivo by inducible p53 expression, we demonstrated that NK cells participate in the elimination of senescent tumors. The elimination of senescent tumor cells is dependent on NKG2D. Interestingly, p53 restoration neither increases ligand expression nor increases the sensitivity to lysis by NK cells. Instead, p53 restoration caused tumor cells to secrete various chemokines with the potential to recruit NK cells. Antibody-mediated neutralization of CCL2, but not CCL3, CCL4 or CCL5, prevented NK cell recruitment to the senescent tumors and reduced their elimination. Our findings suggest that elimination of senescent tumors by NK cells occurs as a result of the cooperation of signals associated with p53 expression or senescence, which regulate NK cell recruitment, and other signals that induce NKG2D ligand expression on tumor cells.

Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia

Abstract: Fibroblast activation protein (FAP) is a candidate universal target antigen because it has been reported to be selectively expressed in nearly all solid tumors by a subset of immunosuppressive tumor stromal fibroblasts. We verified that 18/18 human tumors of various histologies contained pronounced stromal elements staining strongly for FAP, and hypothesized that targeting tumor stroma with FAP-reactive T cells would inhibit tumor growth in cancer-bearing hosts. T cells genetically engineered with FAP-reactive chimeric antigen receptors (CARs) specifically degranulated and produced effector cytokines upon stimulation with FAP or FAP-expressing cell lines. However, adoptive transfer of FAP-reactive T cells into mice bearing a variety of subcutaneous tumors mediated limited antitumor effects and induced significant cachexia and lethal bone toxicities in two mouse strains. We found that FAP was robustly expressed on PDGFR-α+, Sca-1+ multipotent bone marrow stromal cells (BMSCs) in mice, as well as on well-characterized, clinical-grade multipotent human BMSCs. Accordingly, both mouse and human multipotent BMSCs were recognized by FAP-reactive T cells. The lethal bone toxicity and cachexia observed after cell-based immunotherapy targeting FAP cautions against its use as a universal target. Moreover, the expression of FAP by multipotent BMSCs may point toward the cellular origins of tumor stromal fibroblasts.

MHC class II–dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies

Abstract: Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B–MHC II−/−), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)–specific B cell receptor (BCR; IgHMOG-mem) but cannot secrete antibodies. B–MHC II−/− mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell– and B cell–dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B–MHC II−/− mice. In the absence of antibodies, IgHMOG-mem mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle–like structures were observed in IgHMOG-mem mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell–targeted therapies.

Deletion of Asxl1 results in myelodysplasia and severe developmental defects in vivo

Abstract: Somatic Addition of Sex Combs Like 1 (ASXL1) mutations occur in 10-30% of patients with myeloid malignancies, most commonly in myelodysplastic syndromes (MDSs), and are associated with adverse outcome. Germline ASXL1 mutations occur in patients with Bohring-Opitz syndrome. Here, we show that constitutive loss of Asxl1 results in developmental abnormalities, including anophthalmia, microcephaly, cleft palates, and mandibular malformations. In contrast, hematopoietic-specific deletion of Asxl1 results in progressive, multilineage cytopenias and dysplasia in the context of increased numbers of hematopoietic stem/progenitor cells, characteristic features of human MDS. Serial transplantation of Asxl1-null hematopoietic cells results in a lethal myeloid disorder at a shorter latency than primary Asxl1 knockout (KO) mice. Asxl1 deletion reduces hematopoietic stem cell self-renewal, which is restored by concomitant deletion of Tet2, a gene commonly co-mutated with ASXL1 in MDS patients. Moreover, compound Asxl1/Tet2 deletion results in an MDS phenotype with hastened death compared with single-gene KO mice. Asxl1 loss results in a global reduction of H3K27 trimethylation and dysregulated expression of known regulators of hematopoiesis. RNA-Seq/ChIP-Seq analyses of Asxl1 in hematopoietic cells identify a subset of differentially expressed genes as direct targets of Asxl1. These findings underscore the importance of Asxl1 in Polycomb group function, development, and hematopoiesis.

Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell–mediated deletion

Abstract: Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8+ T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8+ T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8–mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.

Lung-resident tissue macrophages generate Foxp3 + regulatory T cells and promote airway tolerance

Abstract: Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MOs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MOs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MOs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MOs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MOs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma.

Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies

Abstract: Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia

Abstract: Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP+ cells, we find that they reside in most tissues of the adult mouse. FAP+ cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP+ cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP+ stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia.

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A

Abstract: Many solid cancers display cellular hierarchies with self-renewing, tumorigenic stemlike cells, or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies, they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs, we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays, but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines, including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion, and targeting IL-17A signaling impaired CIC growth. Notably, IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer.

Phosphofructokinase deficiency impairs ATP generation, autophagy, and redox balance in rheumatoid arthritis T cells

Abstract: In the HLA class II–associated autoimmune syndrome rheumatoid arthritis (RA), CD4 T cells are critical drivers of pathogenic immunity. We have explored the metabolic activity of RA T cells and its impact on cellular function and fate. Naive CD4 T cells from RA patients failed to metabolize equal amounts of glucose as age-matched control cells, generated less intracellular ATP, and were apoptosis-susceptible. The defect was attributed to insufficient induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a regulatory and rate-limiting glycolytic enzyme known to cause the Warburg effect. Forced overexpression of PFKFB3 in RA T cells restored glycolytic flux and protected cells from excessive apoptosis. Hypoglycolytic RA T cells diverted glucose toward the pentose phosphate pathway, generated more NADPH, and consumed intracellular reactive oxygen species (ROS). PFKFB3 deficiency also constrained the ability of RA T cells to resort to autophagy as an alternative means to provide energy and biosynthetic precursor molecules. PFKFB3 silencing and overexpression identified a novel extraglycolytic role of the enzyme in autophagy regulation. In essence, T cells in RA patients, even those in a naive state, are metabolically reprogrammed with insufficient up-regulation of the glycolytic activator PFKFB3, rendering them energy-deprived, ROS- and autophagy-deficient, apoptosis-sensitive, and prone to undergo senescence.

Similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ–resident dendritic cells

Abstract: Dendritic cells (DCs) represent a heterogeneous population of antigen-presenting cells that initiate and orient immune responses in secondary lymphoid organs. In mice, lymphoid organ–resident CD8+ DCs are specialized at cross-presentation and have developed specific adaptations of their endocytic pathway (high pH, low degradation, and high export to the cytosol). In humans, blood BDCA3+ DCs were recently shown to be the homologues of mouse CD8+ DCs. They were also proposed to cross-present antigens more efficiently than other blood DC subsets after in vitro activation, suggesting that in humans cross-presentation is restricted to certain DC subsets. The DCs that cross-present antigen physiologically, however, are the ones present in lymphoid organs. Here, we show that freshly isolated tonsil-resident BDCA1+ DCs, BDCA3+ DCs, and pDCs all cross-present soluble antigen efficiently, as compared to macrophages, in the absence of activation. In addition, BDCA1+ and BDCA3+ DCs display similar phagosomal pH and similar production of reactive oxygen species in their phagosomes. All three DC subsets, in contrast to macrophages, also efficiently export internalized proteins to the cytosol. We conclude that all freshly isolated lymphoid organ–resident human DCs, but not macrophages, display high intrinsic cross-presentation capacity.

Retinoic acid expression associates with enhanced IL-22 production by γδ T cells and innate lymphoid cells and attenuation of intestinal inflammation

Abstract: Retinoic acid (RA), a vitamin A metabolite, modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition, RA treatment enhanced production of the IL-22–responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti–IL-22 neutralizing antibody, demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved, we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells.

Genetic lesions associated with chronic lymphocytic leukemia transformation to Richter syndrome

Abstract: Richter syndrome (RS) derives from the rare transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most commonly of the diffuse large B cell lymphoma (DLBCL) type. The molecular pathogenesis of RS is only partially understood. By combining whole-exome sequencing and copy-number analysis of 9 CLL-RS pairs and of an extended panel of 43 RS cases, we show that this aggressive disease typically arises from the predominant CLL clone by acquiring an average of ∼20 genetic lesions/case. RS lesions are heterogeneous in terms of load and spectrum among patients, and include those involved in CLL progression and chemorefractoriness (TP53 disruption and NOTCH1 activation) as well as some not previously implicated in CLL or RS pathogenesis. In particular, disruption of the CDKN2A/B cell cycle regulator is associated with ∼30% of RS cases. Finally, we report that the genomic landscape of RS is significantly different from that of de novo DLBCL, suggesting that they represent distinct disease entities. These results provide insights into RS pathogenesis, and identify dysregulated pathways of potential diagnostic and therapeutic relevance.

Transient regulatory T cell ablation deters oncogene-driven breast cancer and enhances radiotherapy

Abstract: Rational combinatorial therapeutic strategies have proven beneficial for the management of cancer. Recent success of checkpoint blockade in highly immunogenic tumors has renewed interest in immunotherapy. Regulatory T (T reg) cells densely populate solid tumors, which may promote progression through suppressing anti-tumor immune responses. We investigated the role of T reg cells in murine mammary carcinogenesis using an orthotopic, polyoma middle-T antigen-driven model in Foxp3DTR knockin mice. T reg cell ablation resulted in significant determent of primary and metastatic tumor progression. Importantly, short-term ablation of T reg cells in advanced spontaneous tumors led to extensive apoptotic tumor cell death. This anti-tumor activity was dependent on IFN-γ and CD4+ T cells but not on NK or CD8+ T cells. Combination of T reg cell ablation with CTLA-4 or PD-1/PD-L1 blockade did not affect tumor growth or improve the therapeutic effect attained by T reg cell ablation alone. However, T reg cell targeting jointly with tumor irradiation significantly reduced tumor burden and improved overall survival. Together, our results demonstrate a major tumor-promoting role of T reg cells in an autochthonous model of tumorigenesis, and they reveal the potential therapeutic value of combining transient T reg cell ablation with radiotherapy for the management of poorly immunogenic, aggressive malignancies.

Germinal center B cells govern their own fate via antibody feedback

Abstract: Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells’ own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.

A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation

Abstract: Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.