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Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in murine myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity FcγRI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity., Competing Interests: LF, MN, AZ, AK, NS, AR, AG, AG, LB, TG, AA, AB No competing interests declared, DR This paper was funded by Gilboa Therapeutics where Diana Rasoulouniriana is a shareholder, PR This paper was funded by Gilboa Therapeutics where Peleg Rider is a shareholder, YC This paper was funded by Gilboa Therapeutics where Yaron Carmi is a shareholder, (© 2023, Farhat-Younis et al.)

The pioneering design of chimeric antigen receptor (CAR) T-cell therapy demonstrated the potential of reprogramming the immune system. Nonetheless, T-cell exhaustion, toxicity, and suppressive microenvironments limit their efficacy in solid tumors. We previously characterized a subset of tumor-infiltrating CD4+ T cells expressing the FcγRI receptor. Herein, we detail engineering of a receptor, based on the FcγRI structure, allowing T cells to target tumor cells using antibody intermediates. These T cells showed effective and specific cytotoxicity only when an appropriate antibody was added. Only target-bound antibodies activated these cells, while free antibodies were internalized without activation. Their cytotoxic activity was correlated to target protein density, therefore targeting tumor cells with high antigen density while sparing normal cells with low or no expression. This activation mechanism prevented premature exhaustion. Furthermore, during antibody-dependent cytotoxicity these cells secreted attenuated cytokine levels compared with CAR T cells, thereby enhancing their safety profile. These cells eradicated established melanomas, infiltrated the tumor microenvironment, and facilitated host immune cell recruitment in immunocompetent mice. In NOD/SCID gamma mice the cells infiltrate, persist, and eradicate tumors. As opposed to CAR T-cell therapies, which require changing the receptor across different types of cancer, our engineered T cells remain the same across tumor types, while only the injected antibody changes. Overall, we generated a highly flexible T-cell therapy capable of binding a wide range of tumor cells with high affinity, while preserving the cytotoxic specificity only to cells expressing high density of tumor-associated antigens and using a single manufacturing process., (©2023 The Authors; Published by the American Association for Cancer Research.)

Despite the remarkable successes of cancer immunotherapies, the majority of patients will experience only partial response followed by relapse of resistant tumors. While treatment resistance has frequently been attributed to clonal selection and immunoediting, comparisons of paired primary and relapsed tumors in melanoma and breast cancers indicate that they share the majority of clones. Here, we demonstrate in both mouse models and clinical human samples that tumor cells evade immunotherapy by generating unique transient cell-in-cell structures, which are resistant to killing by T cells and chemotherapies. While the outer cells in this cell-in-cell formation are often killed by reactive T cells, the inner cells remain intact and disseminate into single tumor cells once T cells are no longer present. This formation is mediated predominantly by IFNγ-activated T cells, which subsequently induce phosphorylation of the transcription factors signal transducer and activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in tumor cells. Indeed, inhibiting these factors prior to immunotherapy significantly improves its therapeutic efficacy. Overall, this work highlights a currently insurmountable limitation of immunotherapy and reveals a previously unknown resistance mechanism which enables tumor cells to survive immune-mediated killing without altering their immunogenicity., Competing Interests: AG, NS, LF, DR, AM, CL, JC, KP, NS, GS, AG, RP, CL, MF, DH, VZ, OZ, SK, WH, RD, NR, PR, YC No competing interests declared, (© 2022, Gutwillig et al.)

The recent success of checkpoint blockade therapies has established immunotherapy as one of the most promising treatments for melanoma. Nonetheless, a complete curative response following immunotherapy is observed only in a fraction of patients. To identify what factors limit the efficacy of immunotherapies, we established mouse models that cease to respond to immunotherapies once their tumors exceed a certain stage. Analysis of the immune systems of the organisms revealed that the numbers of tumor-infiltrating dendritic cells (TIDC) drastically decreased with time. Further, in contrast to the current paradigm, once melanoma was established, TIDC did not migrate into sentinel lymph nodes. Instead, they underwent local cell death due to excessive phagocytosis of lysosomes. Importantly, TIDC were required to license the cytotoxic activity of tumor CD8 + T cells, and in their absence, T cells did not lyse melanoma cells. Our results offer a paradigm shift regarding the role of TIDC and a framework to increase the efficacy of immunotherapies. SIGNIFICANCE: This work redefines the role of monocyte-derived dendritic cells in melanoma and provides a novel strategy to increase the efficacy of T-cell-based immunotherapies in nonresponding individuals. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1942/F1.large.jpg., (©2020 American Association for Cancer Research.)

While a high frequency of Th1 cells in tumors is associated with improved cancer prognosis, this benefit has been attributed mainly to support of cytotoxic activity of CD8+ T cells. By attempting to potentiate antibody-driven immunity, we found a remarkable synergy between CD4+ T cells and tumor-binding antibodies. This surprising synergy was mediated by a small subset of tumor-infiltrating CD4+ T cells that express the high-affinity Fcγ receptor for IgG (FcγRI) in both mouse and human patients. These cells efficiently lyse tumor cells coated with antibodies through concomitant crosslinking of their T cell receptor (TCR) and FcγRI. By expressing FcγRI and its signaling chain in conventional CD4+ T cells, we successfully employed this mechanism to treat established solid cancers. Overall, this discovery sheds new light on the biology of this T cell subset, their function during tumor immunity, and the means to utilize their unique killing signals in immunotherapy.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Black band disease (BBD), characterized by a black mat or line that migrates across a coral colony leaving behind it a bare skeleton, is a persistent disease affecting massive corals worldwide. Previous microscopic and molecular examination of this disease in faviid corals from the Gulf of Eilat revealed a number of possible pathogens with the most prominent being a cyanobacterium identified as Pseudoscillatoria coralii. We examined diseased coral colonies using histopathological and molecular methods in order to further assess the possible role of this cyanobacterium, its mode of entry, and pathological effects on the coral host tissues. Affected areas of colonies with BBD were sampled for examination using both light and transmission electron microscopies. Results showed that this dominant cyanobacterium was found on the coral surface, at the coral-skeletal interface, and invading the polyp tissues and gastrovascular cavity. Although tissues surrounding the invasive cyanobacterial filaments did not show gross morphological alterations, microscopic examination revealed that the coral cells surrounding the lesion were dissociated, necrotic, and highly vacuolated. No amoebocytes were evident in the mesoglea of affected tissues suggesting a possible repression of the coral immune response. Morphological and molecular similarity of the previously isolated BBD-associated cyanobacterium P. coralii to the current samples strengthens the premise that this species is involved in the disease in this coral. These results indicate that the cyanobacteria may play a pivotal role in this disease and that the mode of entry may be via ingestion, penetrating the coral via the gastrodermis, as well as through the skeletal-tissue interface.

The surfaces of massive corals of the genus Favia from Eilat, Red Sea, and from Heron Island, Great Barrier Reef, are covered by a layer of eukaryotic microorganisms. These microorganisms are embedded in the coral mucus and tissue. In the Gulf of Eilat, the prevalence of corals covered by patches of eukaryotic microorganisms was positively correlated with a decrease in water temperatures (from 25-28 degrees C in the summer to 20-23 degrees C in winter). Comparisons carried out using transmission and scanning electron microscopy showed morphological similarities between the microorganisms from the two geographically distant reefs. The microorganisms found on and in the tissues were approximately 5-15 microm in diameter, surrounded by scales in their cell wall, contained a nucleus, and included unique auto-florescent coccoid bodies of approximately 1 mum. Such morphological characters suggested that these microorganisms are stramenopile protists and in particular thraustochytrids. Molecular analysis, carried out using specific primers for stramenopile 18S rRNA genes, revealed that 90% (111/123) of the clones in the gene libraries were from the Thraustochytriidae. The dominant genera in this family were Aplanochytrium sp., Thraustochytrium sp., and Labyrinthuloides sp. Ten stramenopile strains were isolated and cultured from the corals. Some strains showed > or =97% similarity to clones derived from libraries of mucus-associated microorganisms retrieved directly from these corals. Fatty acid characterization of one of the prevalent strains revealed a high percentage of polyunsaturated fatty acids, including omega-3. The possible association of these stramenopiles in the coral holobiont appeared to be a positive one.

Recent evidence suggests that there is a dynamic microbial biota living on the surface and in the mucus layer of many hermatypic coral species that plays an essential role in coral well-being. Most of the studies published to date emphasize the importance of prokaryotic communities associated with the coral mucus in coral health and disease. In this study, we report the presence of a protist (Fng1) in the mucus of the hermatypic coral Fungia granulosa from the Gulf of Eilat. This protist was identified morphologically and molecularly as belonging to the family Thraustochytridae (phylum Stramenopile, order Labyrinthulida), a group of heterotrophs widely distributed in the marine environment. Morphological examination of this strain revealed a nonmotile organism c. 35 mum in diameter, which is able to thrive on carbon-deprived media, and whose growth and morphology are inoculum dependent. Its fatty acid production profile revealed an array of polyunsaturated fatty acids. A similar protist was also isolated from the mucus of the coral Favia sp. In light of these findings, its possible contribution to the coral holobiont is discussed.