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Normalized Gene Expression Values from FACS-isolated Lin- FSC low, CD144+ CD31+ clones.

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Clonal analysis results from 20 representative blood vessels 180 and 270 days after tamoxifen exposure show a uniform distribution in clonality within endothelium over the time period. We employed a Chi-squared test to evaluate the null hypothesis of equal distribution, which could not be rejected.

Flow cytometry plots from wild-type mice reconstituted with cyan-fluorescent-protein hematopoietic stem cells CFP-HSCs showing the absence of CFP-HSC derived cells in blood vessels. (a) Flow cytometry plots showing gating of B16 tumors after enzymatic digestion. (b) Within the high expressing CD31 and CD144 subset, we find no contribution from CFP-HSCs (lower panel). (c) In contrast, within the CD45+ CD31-VeCad- subset representing the hematopoietic component of the tumor stroma, progeny of CFP-HSCs are detected. (d) Histologic section of bone marrow showing full blood chimerism 3 months after transplant.

BackgroundActivated T cells have limited antigen presenting capability due to inefficient capture.1 This process can be enhanced through novel chimeric engulfment receptors (CERs) expressing a human Tim-4 phagocyte receptor that recognizes phosphatidylserine (Ptd-Ser)2 fused to T cell and macrophage/dendritic cell-derived signaling domains. CERs can facilitate antigen capture, processing, and presentation, and impart target-dependent cytotoxic function when expressed in T cells. This combined function is hypothesized to improve tumor clearance and durability of response, making CER T cell products ideal clinical candidates.MethodsWe generated Tim-4 receptors fused to toll-like receptor (TLR)-2 or -8, CD28 or CD3 zeta and tested phagocytic, antigen presentation and cytotoxic function in healthy donor T cells. To assess phagocytosis, target cells treated with a small molecule to induce Ptd-Ser externalization were labeled with pH-Rodo followed by co-culture with CER T cells. Activated CER T cells were evaluated by transmission electron microscopy (TEM) or flow cytometry (FC) for lysosomal uptake of cell fragments. Antigen capture and presentation were characterized by FC for the capacity of human papilloma virus 16 (HPV 16) E7 peptide-pulsed CER T cells to activate and induce proliferation of autologous HPV 16 E7-TCR transduced T cells. Cytotoxic function was evaluated in co-culture assays of CER T cells in the presence of subtherapeutic doses of BTKi (ibrutinib)-treated JeKo-1 lymphoma cells.ResultsTEM imaging demonstrated that CER T cells engulfed target cell fragments, illustrated by multi-vesicular bodies containing tumor fragments (some measuring >0.5 uM) and pseudo-pod like formations around apoptotic target cell blebs. RNA analysis revealed upregulation of TLR, myeloid differentiation, and antigen presentation pathways. In the HPV 16 E7 co-culture model, T-cell surface activation markers CD25 and CD69 were upregulated 41% and 23%, respectively, on E7-TCR-T cells relative to controls. In addition, the percentage of dividing E7-TCR-T cells was increased (44% vs 8%) after 6 days in co-culture. Addition of CER T cells to JeKo- 1 target cells in the presence of BTKi at low effector: target ratios enhanced cytotoxicity by over 99%, demonstrating synergy with a targeted small molecule to fully eliminate lymphoma cells.ConclusionsNovel Tim-4/TLR containing CERs can capture tumor cell fragments and present soluble antigen, a function previously demonstrated to be a barrier to effective antigen presentation in T cells. Enhanced T-cell antigen capture and presentation capability alongside inducible and target-specific cytotoxic function in single T cells represents a significant advancement in the potential for chimeric receptor-based therapies.ReferencesLanzavecchia A, Roosnek E, Gregory T, Berman P, Abrignani S. T cells can present antigens such as HIV gp120 targeted to their own surface molecules. Nature 1988 Aug 11;334(6182):530–2.Caronni N, Piperno GM, Simoncello F, Romano O, Vodret S, Yanagihashi Y, et al. TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses. Nat Commun 2021 Apr 14;12(1):2237.

Haematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are haematopoietic stem cells (HSCs), which are multipotent and self-renewing, and generate the entire repertoire of blood and immune cells throughout an animal's life1. Although there have been comprehensive studies on self-renewal, differentiation, physiological regulation and niche occupation in vertebrate HSCs, relatively little is known about the evolutionary origin and niches of these cells. Here we describe the haematopoietic system of Botryllus schlosseri, a colonial tunicate that has a vasculature and circulating blood cells, and interesting stem-cell biology and immunity characteristics2-8. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other3,4,7. Using flow cytometry, whole-transcriptome sequencing of defined cell populations and diverse functional assays, we identify HSCs, progenitors, immune effector cells and an HSC niche, and demonstrate that self-recognition inhibits allospecific cytotoxic reactions. Our results show that HSC and myeloid lineage immune cells emerged in a common ancestor of tunicates and vertebrates, and also suggest that haematopoietic bone marrow and the B. schlosseri endostyle niche evolved from a common origin.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile., It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 “don’t eat me” signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis. Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.

In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 × 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

SummaryHematopoiesis is an essential process that evolved in multicellular animals. At the heart of this process are hematopoietic stem cells (HSCs), which are multipotent, self-renewing and generate the entire repertoire of blood and immune cells throughout life. Here we studied the hematopoietic system of Botryllus schlosseri, a colonial tunicate that has vasculature, circulating blood cells, and interesting characteristics of stem cell biology and immunity. Self-recognition between genetically compatible B. schlosseri colonies leads to the formation of natural parabionts with shared circulation, whereas incompatible colonies reject each other. Using flow-cytometry, whole-transcriptome sequencing of defined cell populations, and diverse functional assays, we identified HSCs, progenitors, immune-effector cells, the HSC niche, and demonstrated that self-recognition inhibits cytotoxic reaction. Our study implies that the HSC and myeloid lineages emerged in a common ancestor of tunicates and vertebrates and suggests that hematopoietic bone marrow and the B. schlosseri endostyle niche evolved from the same origin.

A Gene for Early Acceptance One of the fundamental properties of the immune system is the ability to distinguish self- from nonself–histocompatibility. To gain insight into the evolution and molecular basis of histocompatibility, Voskoboynik et al. (p. 384 ) sought to determine the genetic basis for a natural transplantation reaction that occurs in Botryllus schlosseri , a colonial urochordate. Compatibility allows vascular fusion among individuals, whereas incompatibility results in an inflammatory rejection response. A single gene determined the outcome of the reaction. Like histocompatibility genes in higher organisms, this gene is polymorphic and is expressed in the tissues that participate in the transplantation reaction.

Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies. Cancer Res; 76(6); 1348–53. ©2015 AACR.

Most candidate HIV vaccines are directed at priming memory T cell responses and are being evaluated on their effects on post acquisition viremia and/or disease progression. These vaccines are being studied in areas of high HIV-1 prevalence. As such, we evaluated the frequency of CD4+ T cell decline and time course of opportunistic infections of patients presenting at a major metropolitan hospital in Lima, Peru, an area where such candidate vaccines are being tested. We examined 92 patients with untreated HIV-1 in calendar year 2002: 35% presented with CD4+ T cell counts of200, 25% between 201 and 400, and 17% with400 cells/mm3, 30 of 92 patients presented with overt AIDS, 6 were without an AIDS defining OI but CD4 counts200. Over the course of follow-up, CD4 count decreased by a mean of 31 cells/mm3/year in women and 28 in men (p0.5). Among persons presenting with CD4 counts250 cells/mm3, the median time to first OI was 3.5 years. If clinical endpoints are required to evaluate the clinical effectiveness of T cell based vaccines, extended clinical follow-up of subjects enrolled in such trials will be required.

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI: http://dx.doi.org/10.7554/eLife.00569.001, eLife Digest The tunicates are an evolutionary group that includes species such as sea squirts and sea tulips. Their name comes from the structure known as a ‘tunic’ that surrounds their sac-like bodies. As marine filter feeders, tunicates obtain nutrients by straining food particles from water, and they can live either alone or in colonies depending on the species. Charles Darwin suggested that tunicates may be the key to understanding the evolution of vertebrates, and indeed today they are regarded as the closest living relatives of this group. Colonial tunicates can reproduce either sexually, or asexually by budding. Compatible colonies have the ability to recognize one another and to fuse their blood vessels to form a single organism, whereas incompatible colonies reject one another and remain separate. This recognition process bears some resemblance to the rejection of foreign organ transplants in mammals. Here, Voskoboynik and co-workers present the first genome sequence of a colonial tunicate, Botryllus schlosseri. They used a novel sequencing approach that significantly increased the length of a DNA molecule that can be determined by next-generation sequencing, and allowed large DNA repeat regions to be easily resolved. In total, they sequenced 580 million base pairs of DNA, which they estimate contains roughly 27,000 genes. By comparing the B. schlosseri genome with those of a number of vertebrates, Voskoboynik et al. identified multiple B. schlosseri genes that also participate in the development and functioning of the vertebrate eye, heart, and auditory system, as well as others that may have contributed to the evolution of the immune system and of blood cells. The genome of B. schlosseri thus provides an important new tool for studying the genetic basis of the evolution of vertebrates. DOI: http://dx.doi.org/10.7554/eLife.00569.002

Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover potentiates immune responses against lymphoma cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vitro administration of C2-hIgG1 to murine bone marrow derived macrophages promotes engulfment of apoptotic murine lymphoma cells (38C13 cell line) and ablates the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of pro-inflammatory cytokines tumor necrosis factor (TNF-α) and IL-12p40 to the presence of apoptotic cells. Similarly, uptake of C2-hIgG1 treated lymphoma cells triggers upregulation of the costimulatory markers CD80, CD86, and MHC class II on macrophages and promotes secretion of Th1-recruiting lymphocyte chemokines CXCL9, CXCL10, and CCL5. Accordingly, in vivo administration of C2-hIgG1 partially restores immune responses to dead lymphoma cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not CD19-directed chimeric antigen receptor engineered (CAR-T) T cells. Disclosures No relevant conflicts of interest to declare.

Cell loss by apoptosis is a common feature in tumors. Dying tumor cells induce immune tolerance within the tumor microenvironment largely through highly conserved homeostatic clearance programs that restore tissue immune homeostasis and contribute to the formation of an immunosuppressive niche. The translocation of phosphatidylserine (PS) on cellular membranes, during the initial phases of apoptosis, functions as a recognition and removal signal that limits the immunogenicity of cell death. We examined whether altering clearance of dying cancer cells to elicit inflammatory turnover can allow for and potentiate immune responses against tumor cells. To remove inhibitory signals in the homeostatic clearance pathway we utilized a molecular bridge scaffold to engineer a modified phosphatidylserine bridge protein (FA58C2-hIgG1 or C2-hIgG1) that works as a bridge between apoptotic cells expressing aminophospholipids and phagocytes bearing Fc receptors. In vivo administration of C2-hIgG1 partially restores immune responses to dead tumor cells in antigen cross presentation assays and promotes recruitment and retention of tumor antigen specific CD8+ T cells, dendritic cells, and natural killer cells into tumors. These effects combine to elicit anti-lymphoma immunity, improve responses to immune checkpoint inhibitors, and enhance the effectiveness of adoptive T cell transfers using engineered T Cell Receptors (TCRs) but not chimeric antigen receptor engineered (CAR-T) T cells. Citation Format: Daniel Corey, Aaron Ring, Melissa McCracken, Masanori Miyanishi, Sydney Gordon, Irving Weissman. Super cross-presentation of tumor antigens by synthetic design of an anti-phosphatidylserine bridge protein [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 B051.

B. schlosseri, a colonial marine species, offers a unique platform to study mechanisms underlying cellular recognition. We used adoptive transfer studies, FACs analysis, and RNA-sequencing to study the progression by which fused-colonies eliminate chimeric partners. We show the principle cell type mediating elimination of partners in a chimera is a cytotoxic Morula cell (MC), which utilizes developmental-regulated programmed cell death pathways, initiated during the ‘takeover’ phase of colony life, to eliminate partners. Adoptive transfer of FACs purified cytotoxic MCs from third party donors into recipients recapitulates events of chimeric partner elimination. Transfer of 1×10^5 allogeneic MCs eliminated 33 of 78 (42%) of recipient palleal buds and 20 of 76 (20.5%) adult zooids by day 14 post-transfer. Transfer of phagocytic populations had only minimal effects on recipient colonies. We identified 275 genes (FDR < 0.05) from global RNA-seq expression profiling that increased by expression of at least 50 fold associated with elimination of chimeric partners. These include an IL-17 family member (IL-17C), the phagocytic receptors (MerTK, AXL), and programmed cell removal factors (CALR, MFGE-8). To probe the role of specific receptors we used in vitro studies. Recombinant Il-17c promoted allogeneic cytotoxicity while MFGE-8, a phosphatidylserine binding protein, induced engulfment of apoptotic cells. Studies using chimeric phosphatidylserine binding-cytokine fusion proteins are underway. Preliminary data suggests these fusion proteins promote inflammatory turnover of apoptotic cells. These studies provide new insights by which reverse engineering cell death programs can be utilized to enhance anti-tumor immunity.

We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.

The prolonged effectiveness of antiretroviral therapy (ART) in a developing country is not well established. An observational database was established at the HIV clinic of the Almenara Hospital in Lima Peru in 1996. All 564 initially antiretroviral-naive HIV-infected persons (mean CD4 count of 91 cells/mm3) who received combination ART were followed over time. The overall survival rate was 96% at year 2 94% at year 4 and 91% at year 5. Among persons who initiated therapy with CD4 counts < 100 cells/mm3 the overall survival rate at 3 years was 95%. Opportunistic infections while on ART occurred in 20% of persons. Patients who received 2 reverse transcriptase (RT) inhibitors plus a protease inhibitor had slightly better survival rates and less opportunistic disease in the first year of therapy as compared with those receiving 2 RT inhibitors and a nonnucleoside reverse transcriptase inhibitor or 3 RT inhibitors. This study demonstrates the long-term effectiveness of ART in a developing country urban setting. It provides evidence of the importance of continuing global financing initiatives to provide widespread HIV therapy for countries in the developing world. (authors)