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Supplementary Figure from Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

Supplementary Data from Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses

CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

В работе найден критерий того, что тотальное координатное пространство триномиальной гиперповерхности является гиперповерхностью. Также приведен алгоритм вычисления явного вида кольца Кокса и получены критерии рациональности и факториальности тотального координатного пространства. Библиография: 13 названий.

The therapy of advanced mycosis fungoides (MF) presents a therapeutic challenge, and the search for new therapeutic targets is ongoing. Poly(ADP-ribose) polymerase 1 was shown to be upregulated in patients with advanced MF and could be druggable by a new class of chemotherapeutic agents, PARP-1 inhibitors, which are already in clinical trials for other malignancies; however, the role of PARP-1 inhibitors in MF has never been established. We examined the efficacy of talazoparib in the murine model of cutaneous T-cell lymphoma. The cytotoxic effect of talazoparib on Moloney MuLV-induced T-cell lymphoma (MBL2) cells was a result of G2/M cell cycle arrest via the upregulation of p53. The in vivo experiments confirmed the clinical impact of talazoparib on MF tumors. When talazoparib was combined with the histone deacetylase (HDAC) inhibitor, romidepsin, the cytotoxic effect was synergized via downregulation of the DNA-repair genes Fanconianemia complementation group A (FANCA), Fanconi anemia complementation group D2 (FANCD2), and DNA topoisomerase II binding protein 1(TOPBP1)and stimulation of apoptosis via Blimp-1 (PRDM1)/Bax axis. Romidepsin increased the expression of IRF8 and Bcl-6, leading to upregulation of Blimp1and Bax; whereas talazoparib upregulated Blimp-1 and Bax via upregulation of interferon regulatory factor 4 (IRF4), leading to cleavage of caspases 6 and 7. Thus, a combination of talazoparib with romidepsin demonstrated the synergistic antilymphoma effect and warranted further investigation in a clinical trial.

Six out of 12 Sézary patients shared one clonotype (TRAV13-1*01-TRAJ49*01-TRBV20-1*01-TRBJ2-3*01). TRBV20-1*01 (also known as Vb2) that binds toxic shock syndrome toxin-1 was utilized by Sézary cells among half of the cohort, which would be expected for a common unifying origin.

Simple Summary The identification of the events that accompany cancer progression is essential for developing new therapies. We have used mycosis fungoides, the most common type of cutaneous lymphoma, as a model for our study. We have shown that cancer progression is accompanied by the expansion of exhausted immune cells around malignant cells. Those exhausted cells prevent immune activation, blocking cancer clearance by the immune system. Furthermore, we have demonstrated that novel anti-CD47 immunotherapy with mycosis fungoides leads to the reduction of exhausted T cells accompanied by the expansion of NK and CD8+ T cells. These therapeutic benefits of CD47 blockade were further facilitated by interferon-α, which stimulates cytotoxic cells. Thus, we showed that CD47 might serve as an effective therapeutic target in treating mycosis fungoides. Abstract Cancer progression in mycosis fungoides, the most common form of cutaneous T-cell lymphoma, occurs in a predictable, sequential pattern that starts from patches and that evolves to plaques and later to tumors. Therefore, unlocking the relationship between the microarchitecture of mycosis fungoides and the clinical counterparts of that microstructure represents important steps for the design of targeted therapies. Using multispectral fluorescent imaging, we show that the progression of mycosis fungoides from plaque to tumor parallels the cutaneous expansion of the malignant CD4+ T cells that express TOX. The density of exhausted BTLA+ CD4+ T cells around malignant CD4+TOX+ cells was higher in tumors than it was in plaques, suggesting that undesired safeguards are in place within the tumor microenvironment that prevent immune activation and subsequent cancer eradication. Overriding the CD47 checkpoint with an intralesional SIRPαFc fusion decoy receptor induced the resolution of mycosis fungoides in patients that paralleled an amplified expansion of NK and CD8+ T cells in addition to a reduction of the exhausted BTLA+ CD4+ T cells that were engaged in promiscuous intercellular interactions. These therapeutic benefits of the CD47 blockade were further unleashed by adjuvant interferon-α, which stimulates cytotoxic cells, underscoring the importance of an inflamed microenvironment in facilitating the response to immunotherapy. Collectively, these findings support CD47 as a therapeutic target in treating mycosis fungoides and demonstrate a synergistic role of interferon-α in exploiting these clinical benefits.

The ultraviolet B radiation (UVB) causes skin inflammation, which contributes to the causality and the exacerbation of a number of cutaneous diseases. However, the mechanism of UVB-driven inflammation in the skin remains poorly understood. We show that ferroptosis, a non-apoptotic programmed cell death pathway that is promoted by an excessive phospholipid peroxidation, is activated in the epidermal keratinocytes after their exposure to UVB. The susceptibility of the keratinocytes to UVB-induced ferroptosis depends on the extent of pro-ferroptosis death signal generation and the dysregulation of the glutathione system. Inhibition of ferroptosis prevents the release of HMGB1 from the human epidermal keratinocytes, and blocks necroinflammation in the UVB-irradiated mouse skin. We show that while apoptosis and pyroptosis are also detectable in the keratinocytes after UVB exposure, ferroptosis plays a significant role in initiating UVB-induced inflammation in the skin. Our results have important implications for the prevention and the treatment of a broad range of skin diseases which are fostered by UVB-induced inflammation.

Schwann cells are the principal glial cells of the peripheral nervous system which maintain neuronal homeostasis. Schwann cells support peripheral nerve functions and play a critical role in many pathological processes including injury-induced nerve repair, neurodegenerative diseases, infections, neuropathic pain and cancer. Schwann cells are implicated in a wide range of diseases due, in part, to their ability to interact and modulate immune cells. We discuss the accumulating examples of how Schwann cell regulation of the immune system initiates and facilitates the progression of various diseases. Furthermore, we highlight how Schwann cells may orchestrate an immunosuppressive tumor microenvironment by polarizing and modulating the activity of the dendritic cells.

The tumor microenvironment has been compared with a nonhealing wound involving a complex interaction between multiple cell types. Schwann cells, the key regulators of peripheral nerve repair, have recently been shown to directly affect nonneural wound healing. Their role in cancer progression, however, has been largely limited to neuropathic pain and perineural invasion. In this study, we showed that melanoma activated otherwise dormant functions of Schwann cells aimed at nerve regeneration and wound healing. Such reprogramming of Schwann cells into repair-like cells occurred during the destruction and displacement of neurons as the tumor expanded and via direct signaling from melanoma cells to Schwann cells, resulting in activation of the nerve injury response. Melanoma-activated Schwann cells significantly altered the microenvironment through their modulation of the immune system and the extracellular matrix in a way that promoted melanoma growth in vitro and in vivo. Local inhibition of Schwann cell activity following cutaneous sensory nerve transection in melanoma orthotopic models significantly decreased the rate of tumor growth. Tumor-associated Schwann cells, therefore, can have a significant protumorigenic effect and may present a novel target for cancer therapy.Significance:These findings reveal a role of the nerve injury response, particularly through functions of activated Schwann cells, in promoting melanoma growth.

CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma, has limited treatment options and rare occurrences of long-term remission, thus warranting research into new treatment approaches. CD47 has emerged as a promising target for multiple tumor types, but its role in SS remains unknown. Here, we show that CD47 is highly expressed on Sézary cells in the peripheral blood and skin, and the high level of CD47 expression correlates with worse overall survival (OS) in patients with SS. We also demonstrate that CD47 expression on Sézary cells is under the influence of interleukin 4 (IL-4), IL-7, and IL-13. Signal regulatory protein αFc (SIRPαFc; TTI-621), a novel CD47 decoy receptor, triggers macrophage-mediated phagocytosis of Sézary cells and, when administered in clinical trial settings, results in significant tumor load reduction. We conclude that inhibition of the CD47-SIRPα signaling pathway has therapeutic benefit for patients with SS. This trial was registered at www.clinicaltrials.gov as #NCT02663518.

We find polyhedral divisors corresponding to the torus action of complexity one on affine trinomial hypersurfaces. Explicit computations for particular classes of such hypersurfaces including Pham-Brieskorn surfaces and rational trinomial hypersurfaces are given.

Cutaneous peripheral T-cell lymphoma, not otherwise specified represents a "waste basket" of all cases that cannot be put into another of the categories of mature cutaneous T-cell lymphoma. Previously, the sudden multifocal development of cutaneous CD4 tumors without preceding a patch or plaque stage was classified as d'emblee form of mycosis fungoides (MF). Currently, the term "MF" reserved only for the classic Alibert-Bazin type characterized by the evolution of patches, plaques, and tumors or for variants showing a similar clinical course. The authors describe a 75-year-old white woman who presented with a solitary skin tumor in the right supraclavicular region, with no lymph node or systemic involvement. Local external beam radiation treatment resulted in a complete response. The patient relapsed after 5 months with new tumors in the left neck and left upper chest. Biopsy of the lesions showed a dermal infiltrate of atypical small- to medium-sized T-lymphocytes, and immunohistochemical staining showed coexpression of CD4/CD8 in a subset of these cells, which was confirmed with flow cytometry of the tumor. Although the patient had no preceding patch or plaque stage, the authors herein report this extremely rare case of CD4/CD8 dual-positive peripheral T-cell lymphoma, not otherwise specified presented as MF d'emblee and discuss the seldom similar cases published previously.

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria ( Wigglesworthia glossinidia and Sodalis glossinidius ) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia , trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.

Paratransgenesis, a strategy for control of certain arthropod-borne infectious diseases, involves expression of disease transmission-blocking molecules by genetically altered symbiotic bacteria of insect vectors. Field use of this approach relies on a method for dispersal of genetically transformed bacteria to their host insects. Rhodococcus rhodnii , a symbiotic bacterium of the Chagas disease vector, Rhodnius prolixus (Stal), has been transformed genetically to express several molecules. Here we report a strategy for dispersal of genetically altered R. rhodnii to target populations of R. prolixus that uses a synthetic substrate termed CRUZIGARD. This preparation mimics natural feces of R. prolixus and, when impregnated with genetically altered symbiotic bacteria, simulates the natural coprophagic method of symbiont dispersal used by the reduviid bug. CRUZIGARD laden with genetically altered symbionts was applied monthly to closed cages of R. prolixus nymphs. Uptake and retention of the recombinant bacteria was demonstrated under laboratory conditions and simulated field conditions in which competing environmental microbes were present. In the simulated field study conducted in Guatemala, nearly 50% of R. prolixus nymphs exposed to bacteria-laden CRUZIGARD retained the transgenic microbes throughout their development. In these paratransgenic insects, the genetically altered symbionts comprised nearly 95% of the bacterial colony forming units. No adverse effects of CRUZIGARD on insect development and fecundity were noted. CRUZIGARD represents a potentially powerful method for dispersal of recombinant symbiotic bacteria among field populations of the Chagas disease vector, R. prolixus .

Infection often triggers an exacerbation of chronic ITP. The precise mechanism for the decreased platelet count associated with infection is not completely understood. However, interference with megakaryopoiesis is often invoked, and increased production of autoantibodies is also possible. We hypothesized that stimulation of innate immunity could enhance phagocytosis in the reticuloendothelial system and thus contribute to an exacerbation of chronic ITP in the setting of infection. The innate immune response involves activation of Toll-like receptors (TLRs) that initiate intracellular signaling through MyD88 kinase as well as through other incompletely characterized MyD88-independent pathways. We have previously shown that Syk is a crucial signaling tyrosine kinase involved in Fcγ receptor-mediated platelet phagocytosis and that Syk-specific dsRNA inhibits antibody-mediated platelet phagocytosis. Furthermore, the ligand binding requirements for Syk are not stringently limited to classical tandem immunoreceptor tyrosine-based activation motifs. We speculated that TLR4 might employ Syk tyrosine kinase intracellular signaling in human macrophages as a mechanism to enhance immune platelet phagocytosis. The present study was undertaken to address the following questions: Does lipopolysaccharide (LPS) stimulation, an in vitro model for innate immunity triggered by gram-negative bacterial infection, augment antibody-dependent phagocytosis of platelets? Does TLR4 associate with Syk tyrosine kinase in macrophages? Does LPS stimulation induce phosphorylation of Syk? Methods: Antibody-mediated phagocytosis of platelets by THP-1 (human macrophage) cells in culture was assayed by co-incubation of THP-1 cells with or without LPS stimulation at 37°C with platelets that were labeled with 5-(and 6-) carboxyfluorescein diacetate mixed isomers (CFDA) in the presence of human serum containing antihuman GPIIIa IgG antibody. Association of TLR4 with the tyrosine kinase, Syk, was studied in THP-1 cells by immunoprecipitation and immunoblotting after ligation with anti-TLR4 monoclonal antibody (mAb) or stimulation with LPS. Phosphorylation of TLR4-associated Syk after LPS stimulation was studied by immunoprecipitation and immunodetection with anti-TLR4, antiphosphotyrosine (PY99), and anti-Syk mAbs. Results: Antibody-mediated platelet phagocytosis by THP-1 cells was enhanced by 50% after LPS exposure. In five experiments a mean of 21.8% ± 4.1% CFDA-labeled platelets were ingested by THP-1 cells upon antiplatelet antibody exposure in the absence of LPS, while 32.8% ± 6.3% were phagocytosed after LPS (p Conclusions: These studies support the hypothesis that activation of TLR4 in the setting of infection contributes to enhanced antibody-mediated platelet phagocytosis by macrophages and thus exacerbates chronic ITP. Syk becomes phosphorylated upon TLR4 stimulation of macrophages and may play a role in signal transduction triggered by the innate immune system through TLR4. These novel findings may have an impact on the understanding and management of immune thrombocytopenia in the setting of infectious diseases and offer a new therapeutic target for chronic ITP.

We have previously shown that Syk-specific siRNA inhibits FcγRIIA mediated platelet phagocytosis in the human macrophage cell line, THP-1. Surprisingly, we observed that THP-1 cell-cell interactions were affected by Syk-specific siRNA suggesting that Syk may be important in the signaling pathway for physiological cell spreading. As reported by Brown et al. (Nature418:200, 2002), homophilic ligation of PECAM-1 on macrophages triggers cell-cell detachment, prompting us to speculate that Syk may be involved in PECAM-1 mediated signaling. PECAM-1 is a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine inhibitory motif (ITIM) required for inhibitory signaling and cell spreading via binding protein tyrosine phosphatase. The structure of Syk reveals considerable flexibility in the relative orientation of its tandem SH2 domains providing a molecular basis for the potential involvement of Syk in a variety of signal transduction pathways. Thus, the five tyrosine residues present in the cytoplasmic domain of human PECAM-1 could serve as docking sites for recruitment of other intracellular signaling molecules such as the protein tyrosine kinase, Syk. The present studies test the hypothesis that Syk may mediate an activation influence on intercellular interaction upon PECAM-1 ligation. METHODS: Association of Syk with PECAM-1 was studied in THP-1 cells by immunoprecipitation and immunoblotting with anti-PECAM-1 monoclonal antibody with or without Syk-specific siRNA. The effect of Syk-specific siRNA on THP-1 cells was evaluated by morphology, cell growth kinetics, Trypan Blue exclusion, annexin V staining, and DNA fragmentation analysis. A series of synthetic peptides including sequences within the ITIM region of PECAM-1 were evaluated for their ability to bind Syk directly in THP-1 lysates and to phosphorylate Syk intracellularly. RESULTS: First, Syk protein was co-immunoprecipitated from THP-1 cell lysates by anti-PECAM-1 monoclonal antibody, and Syk-specific siRNA knocked down PECAM-1 associated Syk in THP-1 cells. Second, knock down of Syk by siRNA resulted in decreased cell spreading and cell proliferation. Trypan Blue exclusion, annexin V staining, and DNA fragmentation were identical in THP-1 cells transfected with control dsRNA and Syk-specific siRNA. Third, the synthetic peptide of residues 658-691 within the ITIM region of PECAM-1 that contains two phosphotyrosines (N-S-D-V-Q-pY-T-E-V-Q-V-S-S-A-E-S-H-K-D-L-G-K-K- D-T-E-T-V-pY-S-E-V-R-K) bound directly to Syk. Fourth, ligation of PECAM-1 on THP-1 cells by anti-PECAM-1 monoclonal antibody resulted in Syk phosphorylation. Fifth, the dual phosphotyrosine 658-691 PECAM-1 peptide triggered direct phosphorylation of Syk in permeabilized THP-1 cells. CONCLUSIONS: Our novel observations suggest that signal transduction after PECAM-1 ligation may trigger an activation pathway through the tyrosine kinase, Syk, and facilitate intercellular adhesion. The decreased proliferation observed in THP-1 cells transfected with Syk-specific siRNA is not a result of cell death or apoptosis. Based on our findings, we propose that PECAM-1 may exhibit both ITIM (inhibitory) and ITAM (activation) properties and that the balance of phosphatase and Syk activity triggered by PECAM-1 homophilic binding may affect the fate of intercellular interaction. These processes are expected to influence the net degree of phagocytosis of opsonized platelets by the reticuloendothelial system in patients with immune thrombocytopenia.