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S-Figure 1. KSHV reactivation by PEP005; S-Figure 2. Chromatin immunoprecipitations; S-Figure 3. Intersection analysis of genes differentially-expressed in response to each treatment in different PEL cell lines.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic double-stranded DNA virus and the etiologic agent of Kaposi’s sarcoma and hyperinflammatory lymphoproliferative disorders. Understanding the mechanism by which KSHV increases the infected cell population is crucial for curing KSHV-associated diseases. Here we demonstrate that KSHV preferentially infects CD14+monocytes and sustains viral replication through the viral interleukin-6 (vIL6)-mediated activation of STAT1 and 3. Using vIL6-sufficient and vIL6-deficient recombinant KSHV, we demonstrated that vIL6 plays a critical role in promoting the proliferation and differentiation of KSHV-infected monocytes into macrophages. The macrophages derived from vIL6-sufficient KSHV infection showed a distinct transcriptional profile of elevated IFN-pathway activation with immune suppression and were compromised in T-cell stimulation function compared to those from vIL6-deficient KSHV infection or uninfected control. These results highlight a clever strategy, in which KSHV utilizes vIL6 to secure its viral pool by expanding infected dysfunctional macrophages. This mechanism also facilitates KSHV to escape from host immune surveillance and to establish a lifelong infection. 160SummaryKSHV causes multiple inflammatory diseases, however, the underlying mechanism is not clear. Shimoda et al. demonstrate that KSHV preferentially infects monocytes and utilizes virally encoded interleukin-6 to expand and deregulate infected monocytes. This helps the virus escape from host immune surveillance.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases., Shimoda et al. present evidence that a small functional peptide derived from the KSHV transactivator could be used to target MYC in cancer. The authors find that the peptide attenuates MYC expression, reduces cell proliferation, and selectively kills cancer cells in tissue culture and a xenograft tumor mouse model, by blocking recruitment of coactivator complexes (Nuclear receptor coactivator 2, p300, and SWI/SNF proteins) to the MYC promoter.

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we identified a small peptide derived from the Kaposi's sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular c-MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (​SLAM seq) with target-transcriptional analyses further confirmed that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may have significant potential as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvβ3 and α5β1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4β1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvβ3, α5β1, and α4β1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR 2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

Membrane-bound oligosaccharides form the interfacial boundary between the cell and its environment, mediating processes such as adhesion and signaling. These structures can undergo dynamic changes in composition and expression based on cell type, external stimuli, and genetic factors. Glycosylation, therefore, is a promising target of therapeutic interventions for presently incurable forms of advanced cancer. Here, we show that cholangiocarcinoma metastasis is characterized by down-regulation of the Golgi α-mannosidase I coding gene MAN1A1, leading to elevation of extended high-mannose glycans with terminating α-1,2-mannose residues. Subsequent reshaping of the glycome by inhibiting α-mannosidase I resulted in significantly higher migratory and invasive capabilities while masking cell surface mannosylation suppressed metastasis-related phenotypes. Exclusive elucidation of differentially expressed membrane glycoproteins and molecular modeling suggested that extended high-mannose glycosylation at the helical domain of transferrin receptor protein 1 promotes conformational changes that improve noncovalent interaction energies and lead to enhancement of cell migration in metastatic cholangiocarcinoma. The results provide support that α-1,2-mannosylated N-glycans present on cancer cell membrane proteins may serve as therapeutic targets for preventing metastasis.

Actinic keratosis (AK) is a precancerous skin lesion that is common in HIV-positive patients. Without effective treatment, AKs can progress to squamous cell carcinoma. Ingenol mebutate, a PKC agonist, is a US Food and Drug Administration–approved (FDA-approved) topical treatment for AKs. It can induce reactivation of latent HIV transcription in CD4(+) T cells both in vitro and ex vivo. Although PKC agonists are known to be potent inducers of HIV expression from latency, their effects in vivo are not known because of the concerns of toxicity. Therefore, we sought to determine the effects of topical ingenol mebutate gel on the HIV transcription profile in HIV-infected individuals with AKs, specifically in the setting of suppressive antiretroviral therapy (ART). We found that AKs cleared following topical application of ingenol mebutate and detected marginal changes in immune activation in the peripheral blood and in skin biopsies. An overall increase in the level of HIV transcription initiation, elongation, and complete transcription was detected only in skin biopsies after the treatment. Our data demonstrate that application of ingenol mebutate to AKs in ART-suppressed HIV-positive patients can effectively cure AKs as well as disrupt HIV latency in the skin tissue microenvironment in vivo without causing massive immune activation.

CD40L plays a major role in immune response and is a major therapeutic target for inflammation. Integrin α5β1 and CD40 simultaneously bind to CD40L. It is unclear if α5β1 and CD40 work together in CD40/CD40L signaling or how α5β1 binds to CD40L. In this article, we describe that the integrin-binding site of human CD40L is predicted to be located in the trimeric interface by docking simulation. Mutations in the predicted integrin-binding site markedly reduced the binding of α5β1 to CD40L. Several CD40L mutants defective in integrin binding were defective in NF-κB activation and B cell activation and suppressed CD40L signaling induced by wild-type CD40L; however, they still bound to CD40. These findings suggest that integrin α5β1 binds to monomeric CD40L through the binding site in the trimeric interface of CD40L, and this plays a critical role in CD40/CD40L signaling. Integrin αvβ3, a widely distributed vascular integrin, bound to CD40L in a KGD-independent manner, suggesting that αvβ3 is a new CD40L receptor. Several missense mutations in CD40L that induce immunodeficiency with hyper-IgM syndrome type 1 (HIGM1) are clustered in the integrin-binding site of the trimeric interface. These HIGM1 CD40L mutants were defective in binding to α5β1 and αvβ3 (but not to CD40), suggesting that the defect in integrin binding may be a causal factor of HIGM1. These findings suggest that α5β1 and αvβ3 bind to the overlapping binding site in the trimeric interface of monomeric CD40L and generate integrin-CD40L-CD40 ternary complex. CD40L mutants defective in integrins have potential as antagonists of CD40/CD40L signaling.

CD40L plays a major role in immune response and is a target for inflammatory disease therapy. Besides CD40, CD40L binds to several integrins but their role in signaling is unclear. We showed that integrins αvβ3 and α5β1 bind to the CD40L trimeric interface through classical ligand binding site of integrins (site 1). Several CD40L mutants from HIGM1 (hyper-IgM syndrome type 1) patients were clustered in trimeric interface and defective in integrin binding, and in NF-κB and B cell activation, but still bound CD40 and acted as antagonists of CD40L signaling. Thus, integrins play a critical role in CD40L signaling. Previous studies showed that CX3CL1 and sPLA2-IIA can activate integrins by binding to a recently identified allosteric site (site 2) of integrins. Using cell-free assays, we found that soluble (s)-CD40L activated soluble integrins, α4β1, αvβ3 and α5β1, and induced their binding to known specific ligands independent of inside-out signaling. Also, sCD40L activated cell-surface integrins in CHO cells lacking CD40. Finally, sCD40L bound to a cyclic peptide derived from site 2. These findings indicate that CD40L acts as an allosteric activator of integrins by binding to site 2. Docking simulation predicted that the integrin site 2-binding site of CD40L is located outside of CD40L trimer. Importantly, four HIGM1 mutations are clustered in the predicted site 2-binding site of CD40L. The K143T and G144E mutants were the most defective in integrin activation, indicating that integrin binding occurs around these residues of the predicted site 2-binding site. We propose that allosteric integrin activation by CD40L plays a role in CD40L signaling and that defective integrin site 2-binding may be causal for defective CD40L signaling in HIGM1.

Memory CD4+ T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4+ T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells.

Background We conducted a phase Ib dose-confirmation study of indoximod with radiation, followed by indoximod with cyclic temozolomide therapy, to evaluate safety and overall survival (OS) in children with newly diagnosed DIPG (diffuse intrinsic pontine glioma). Indoximod is a small-molecule inhibitor of the IDO pathway that reverses immune suppression imposed by tumor microenvironments. DIPG is a uniformly fatal orphan disease with no curative treatment options. Methods Children age 3 to 21 years, with newly diagnosed DIPG were eligible for treatment with oral indoximod (38.4 mg/kg/day divided BID, throughout) combined with fractionated conformal radiation therapy (54 Gy in 30 fractions), followed by cyclic oral chemo-immunotherapy using indoximod combined with temozolomide (200 mg/m2/day, days 1-5 of each 28-day cycle). The indoximod dose was the previously determined recommended phase-II dose (RP2D), and the study design called for phase I monitoring to confirm safety of this dose in DIPG patients. Results Thirteen children (median age 9 years, range 5 to 20 years) with newly diagnosed DIPG were treated using this indoximod-based radio-chemo-immunotherapy regimen. The 12-month OS was 62% (8/13) and estimated median OS was 14.5 months (follow-up range 4.8 to 22 months) with 4 patients remaining in follow-up. This compares favorably to the expected 12-month OS of approximately 45% and median OS of approximately 10.8 months from published historical controls (Kilburn, et al; 2017). Two patients experienced near-complete responses until showing relapse at 7.6 months and 13.3 months of study therapy. Patients were followed with quantitative volumetric analyses of MRIs and serial measurements of peripheral blood inflammatory monocytes, T cell activation, and pro-inflammatory cytokines. The most common adverse events attributed to indoximod were thrombocytopenia, diarrhea, nausea, vomiting, and fatigue. Conclusion Adding indoximod-based immunotherapy to conventional radiation and chemotherapy for up-front therapy of children with DIPG appears to be well tolerated with improved outcomes. Clinical trial identification NLG2105, NCT02502708. Legal entity responsible for the study NewLink Genetics Corporation. Funding NewLink Genetics Corporation, Alex's Lemonade Stand Foundation, Cannonball Kids cancer Foundation, Beloco Foundation, Eli’s Block Party Foundation, Hyundai Hope on Wheels Foundation, Northern Nevada Children’s Cancer Research Foundation, CAM Fund, Press On Foundation. Disclosure T.S. Johnson: Research grant / Funding (institution): NewLink Genetics Corporation. E.P. Kennedy: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: NewLink Genetics Corporation. N. Vahanian: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: NewLink Genetics Corporation. D.H. Munn: Advisory / Consultancy, Shareholder / Stockholder / Stock options, Licensing / Royalties: NewLink Genetics Corporation. All other authors have declared no conflicts of interest.

Numerous studies of relatively few patients have linked T cell receptor (TCR) genes to psoriasis but have yielded dramatically conflicting results. To resolve these discrepancies, we have chosen to mine RNA-Seq datasets for patterns of TCR gene segment usage in psoriasis. A meta-analysis of 3 existing and 1 unpublished datasets revealed a statistically significant link between the relative expression of TRAJ23 and psoriasis and the psoriasis-associated cytokine IL-17A. TRGV5, a TCR-γ segment, was also associated with psoriasis but correlated instead with IL-36A, other IL-36 family members, and IL-17C (not IL-17A). In contrast, TRAJ39 was strongly associated with healthy skin. T cell diversity measurements and analysis of CDR3 sequences were also conducted, revealing no psoriasis-associated public CDR3 sequences. Finally, in comparison with the expression of TCR-αβ genes, the expression of TCR-γδ genes was relatively low but mildly elevated in psoriatic skin. These results have implications for the development of targeted therapies for psoriasis and other autoimmune diseases. Also, the techniques employed in this study have applications in other fields, such as cancer immunology and infectious disease.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

International audience; Aim : The success of invasive species in their introduced range is often assumed to result from evolutionary changes in defence and growth traits, or as a response to more favourable conditions. The latter is assumed particularly for species exhibiting low, or even no, sexual reproduction in the introduced range.Location and Methods : Here, we compared Japanese (native range) and French (introduced range) populations of Fallopia japonica under common growth conditions in a glasshouse. We measured height, aboveground and belowground mass, stem stiffness, leaf toughness and secondary metabolites found in hydroalcoholic extracts of rhizomes of F. japonica, as well as the competitive response of Rubus caesius (a co‐occurring native species in the invaded range) in the presence of F. japonica from both ranges.Results : Aboveground biomass, height, stem stiffness and composition of secondary metabolites were not significantly different between the two ranges, showing that increased aboveground vigour observed in situ in France is probably the result of a plastic response following the release of abiotic or biotic constraints from the native range. On the other hand, belowground mass, effect on R. caesius, and leaf toughness were all higher in French populations, suggesting increases in competitive ability and defence mechanisms. These differences between France and Japan may be explained either by post‐introduction evolution or by the introduction in Europe, in nineteenth century, of an exceptionally vigorous clone (pre‐adaptation).Main conclusions : Our results provide evidence that the high vigour of this major invasive species in its introduced range is probably due to both a response to more favourable conditions and rapid evolution.

Humoral responses to non-proteinaceous antigens (i.e. T cell independent, TI) are a key component of the early response to bacterial and viral infection and a critical driver of systemic autoimmunity. However, mechanisms that regulate TI humoral immunity are poorly defined. Herein we report that B cell-intrinsic induction of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase1 (IDO1), is a key mechanism limiting TI antibody responses. When Ido1−/− mice were immunized with TI antigens, there was a significant increase in antibody titers and formation of extrafollicular antibody secreting cells (ASCs) compared to controls. This effect was specific to TI antigens, as Ido1 disruption did not affect immunoglobulin production after immunization with protein antigens. The effect of IDO1 abrogation was confined to the B cell compartment, as adoptive transfer of Ido1−/− B cells to B cell deficient mice was sufficient to replicate increased TI responses observed in Ido1−/− mice. Moreover, in vitro activation with toll-like receptor ligands or B cell receptor crosslinking rapidly induced Ido1 expression and activity in purified B cells, and Ido1−/− B cells displayed enhanced proliferation and cell survival associated with increased immunoglobulin and cytokine production compared to wild type B cells. Thus, our results demonstrate a novel, B cell intrinsic, role for IDO1 as a regulator of humoral immunity that has implications for both vaccine design and prevention of autoimmunity.

B cells are increasingly regarded as integral to the pathogenesis of multiple sclerosis, in part as a result of the success of B cell–depletion therapy. Multiple B cell–dependent mechanisms contributing to inflammatory demyelination of the CNS have been explored using experimental autoimmune encephalomyelitis (EAE), a CD4 T cell–dependent animal model for multiple sclerosis. Although B cell Ag presentation was suggested to regulate CNS inflammation during EAE, direct evidence that B cells can independently support Ag-specific autoimmune responses by CD4 T cells in EAE is lacking. Using a newly developed murine model of in vivo conditional expression of MHC class II, we reported previously that encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the sole APC. In this study, we find that B cells cooperate with dendritic cells to enhance EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG) immunization. Further, increasing the precursor frequency of MOG-specific B cells, but not the addition of soluble MOG-specific Ab, is sufficient to drive EAE in mice expressing MHCII by B cells alone. These data support a model in which expansion of Ag-specific B cells during CNS autoimmunity amplifies cognate interactions between B and CD4 T cells and have the capacity to independently drive neuroinflammation at later stages of disease.

Herein we will review the role of glycans in determining the functionality and specificity of various components of the immune system. Specific topics covered include: the specific glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated to date. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and serum proteins, especially the site-specific glycosylation patterns of specific antibody classes and subclasses.

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Currently, treatment options for patients with PEL are limited. Oncolytic viruses have been engineered as anticancer agents and have recently shown increased therapeutic promise. Similarly, lytic activation of endogenous viruses from latently infected tumor cells can also be applied as a cancer therapy. In theory, such a therapeutic strategy would induce oncolysis by viral replication, while simultaneously stimulating an immune response to viral lytic cycle antigens. We examined the combination of the FDA-approved drug ingenol-3-angelate (PEP005) with epigenetic drugs as a rational therapeutic approach for KSHV-mediated malignancies. JQ1, a bromodomain and extra terminal (BET) protein inhibitor, in combination with PEP005, not only robustly induced KSHV lytic replication, but also inhibited IL6 production from PEL cells. Using the dosages of these agents that were found to be effective in reactivating HIV (as a means to clear latent virus with highly active antiretroviral therapy), we were able to inhibit PEL growth in vitro and delay tumor growth in a PEL xenograft tumor model. KSHV reactivation was mediated by activation of the NF-κB pathway by PEP005, which led to increased occupancy of RNA polymerase II onto the KSHV genome. RNA-sequencing analysis further revealed cellular targets of PEP005, JQ1, and the synergistic effects of both. Thus, combination of PEP005 with a BET inhibitor may be considered as a rational therapeutic approach for the treatment of PEL. Mol Cancer Ther; 16(11); 2627–38. ©2017 AACR.

Background Pyoderma gangrenosum (PG) is a debilitating ulcerative skin disease that is one of the most common associated diseases seen in patients with inflammatory bowel disease and rheumatoid arthritis. Although PG is classified as a neutrophilic dermatosis, its pathophysiology is poorly understood. Objective Use data obtained from patient-reported histories, immunohistochemistry, and gene expression analysis to formulate a hypothesis on PG pathophysiology. Methods Ten PG patients participated and answered questions about new ulcer formation. Skin biopsies of healed prior ulcers and adjacent normal skin were obtained from four patients for immunohistochemistry. Scars from healthy patients and patients with discoid lupus were used as additional controls. New onset PG papules were analyzed using immunohistochemistry and gene expression analysis via quantitative real-time PCR. Results All PG patients reported that healed sites of previous ulceration are refractory to re-ulceration. Simultaneous biopsies of healed and uninvolved skin triggered ulceration only in the latter. On immunohistochemistry, healed PG scars showed complete loss of pilosebaceous units, which were present in normal skin, and to a lesser extent in control scars, and discoid scars. Early PG papules showed perivascular and peripilosebaceous T cell infiltrates, rather than neutrophils. These early inflammatory events were dominated by increased gene expression of CXCL9, CXCL10, CXCL11, IL-8, IL-17, IFNG, and IL-36G and transcription factors consistent with Th1 phenotype. Limitations Small sample size was the main limitation. Conclusion We put forth the hypothesis that PG is a T cell response resulting in the destruction of pilosebaceous units.

Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T-cell dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH -cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway.

CD103

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central “memory” cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a “memory” or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naïve animals revealed that the HEL-specific clones displayed effector and central “memory” cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a “memory” or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

Fallopia japonica is native to Japan, Taiwan and Korea. It is now widely naturalized in Europe and North America, and is regarded as one of the worst invasive alien species. For Japanese people, F. japonica has been one of the most familiar and useful wild plants for centuries. It is rare to regard the plant as a harmful weed in Japan. Fallopia japonica has many ethnobotanical uses as an edible and medicinal plant. Therefore it was recorded in many historical documents, by different names. A dictionary of local plant names recorded 689 words for F. japonica, and it was the plant with the most names among all plants listed. As a case study of F. japonica, we conducted surveys on its habitats and growth from the foot to the alpine belt of Mt. Fuji, the highest mountain in Japan. Fallopia japonica was found widely in man-made and natural habitats but was restricted to open, sunny sites. The plant was smaller in the habitats where the vegetation is cut repeatedly. Japanese phytosociological studies regarded F. japonica as the character species of an Artemisietea principis and as a constituent species of diverse herbaceous, shrub and open woodland communities. In Japan, vegetation is controlled by cutting or mowing near habitations and other infrastructure. In meadows, tall grasses dominate the vegetation. In mountainous areas, trees are dominant and open areas are limited. It is difficult for F. japonica to overgrow a wide area and to form a large stand. In addition, the plant has been regarded as useful plant. These matters seem to make F. japonica a common and familiar plant, not a pest, for Japanese people.

γδ T cells have been proposed to play a major role in the pathophysiology of psoriasis and other immune-mediated skin diseases. T cell repertoire analysis is one method of characterizing the T cell component of an immune response. Although traditionally conducted on T cell receptor (TCR)-dedicated PCR products, with the appropriate bioinformatics pipeline and sequencing depth, T cell repertoire analysis can also be performed directly on RNA-Seq data sets, allowing for informative correlative studies to be performed in parallel. To compare the role of γδ versus αβ T cells in psoriasis, we have chosen to mine psoriasis and healthy control skin RNA-Seq data sets for TCR receptor sequences. Unlike prior studies, our results are validated in four independently collected datasets. We demonstrate that in the setting of psoriasis, TCRγ and TCR δ-mapped reads are only a small fraction of their TCR α and TCR β counterparts. Although we identified several TCR sequences that were found in multiple skin samples, none were associated with HLA-Cw6. We also observed an increase in TCR repertoire diversity in psoriatic biopsies, demonstrating the polyclonal nature of the T cell infiltrate in psoriasis. Analysis of CDR3 sequences were also conducted revealing no psoriasis-associated public CDR3 sequences. A meta-analysis revealed a statistically significant link between the of TRAJ23 usage in psoriasis and the psoriasis-associated cytokine IL17A. TRGV5, a TCR γ segment, was also associated with psoriasis but correlated instead with IL36A. This work highlights novel strategies for characterizing tissue-infiltrating T cells from RNA-Seq data sets, an approach with broad applications in the fields of autoimmunity and cancer immunotherapy.

CD103+ dendritic cells are critical for cross-presentation of tumor antigens. Here we have shown that during immunotherapy, large numbers of cells expressing CD103 arose in murine tumors via direct differentiation of Ly6c+ monocytic precursors. These Ly6c+CD103+ cells could derive from bone-marrow monocytic progenitors (cMoPs) or from peripheral cells present within the myeloid-derived suppressor cell (MDSC) population. Differentiation was controlled by inflammation-induced activation of the transcription factor p53, which drove upregulation of Batf3 and acquisition of the Ly6c+CD103+ phenotype. Mice with a targeted deletion of p53 in myeloid cells selectively lost the Ly6c+CD103+ population and became unable to respond to multiple forms of immunotherapy and immunogenic chemotherapy. Conversely, increasing p53 expression using a p53-agonist drug caused a sustained increase in Ly6c+CD103+ cells in tumors during immunotherapy, which markedly enhanced the efficacy and duration of response. Thus, p53-driven differentiation of Ly6c+CD103+ monocytic cells represents a potent and previously unrecognized target for immunotherapy.

柑橘園地域でため池の水質が水草種数に及ぼす影響を明らかにするために，松山市内北条地区で，水草のない森林中の5池を含む43のため池を，各池で1回，1998年から2003年の間に8月または9月に調査した。全体で25種の水草が認められたが，1池当りの種数は平均して2.8種であった。水質については，主成分分析により，2つの重要な成分，肥料成分を含む集水域からの主要イオン類の溶脱を反映するPC 1と，CO2の供給と消費を反映するPC 2が認められた。水面または池底を覆う水草の植被率が30%未満の池群（n = 21，水草のない5池を除く）では，1池当りの水草種数がPC 1の得点及び全窒素濃度と有意な負の相関を示した。また，10 g m-3以上の懸濁物質を伴う高栄養塩濃度の下で，沈水植物が消滅する傾向が認められた。栄養塩，特に窒素の濃度の増大は沈水植物を減少させて，1池当りの水草種数を減らすと見られた。他方，植被率が30 % 以上の池群（n = 17）ではこのような明白な関係が認められなかった。この池群ではPC 2が低く，CO2の消費に比べて供給が多かった。高栄養塩濃度下で，両池群は濁水と清水の二者択一的安定状態（alternative stable states）にほぼ該当すると見られた。

Memory B-cell development, maintenance, and differentiation have been believed to be tightly regulated by T cells through major histocompatibility complex (MHC)-II-restricted cognate interaction. However, recent studies have indicated that memory B cells are a heterogeneous population with various ontogeny and with derivations from T-cell-independent as well as T-cell-dependent response. In addition, several studies highlight T-cell-independent maintenance and differentiation of memory B cells by innate polyclonal stimuli associated with Toll-like-receptors (TLRs). Thus, both T-cell-dependent and T-cell-independent mechanisms with possibly different physiological functions are required for maximal memory B-cell maintenance and differentiation. In this review, we discuss the mechanisms of memory B-cell maintenance and differentiation by re-examining the requirement for MHC-II-restricted B-cell antigen presentation based on our recent study in a mouse model that lacks MHC-II on memory B cells. We propose that MHC-II-restricted memory B-cell maintenance and differentiation are the indispensable mechanisms by which we achieve health and the maximum quality of memory response with specific antibody production, while preventing plasma cell differentiation from self-reactive memory B cells under the control of T-helper cells and possibly regulatory T cells.

Although the importance of MHC class II (MHC-II) in acute homeostatic proliferation of regulatory T (Treg) cells has been established, we considered here the maintenance and state of Treg cells in mice that are almost completely devoid of MHC-II in their periphery but still make their own CD4 T cells and Treg cells. The latter was accomplished by conditional deletion of a loxP-flanked MHC-II β-chain allele using a TIE2Cre transgene, which causes a very high degree of deletion in hemopoietic/endothelial progenitor cells but without deletion among thymic epithelial cells. Such conditional MHC-II-deficient mice possess their own relatively stable levels of CD4+CD25+ cells, with a normal fraction of Foxp3+ Treg cells therein, but at a level ∼2-fold lower than in control mice. Thus, both Foxp3low/− CD4+CD25+ cells, said to be a major source of IL-2, and IL-2-dependent Foxp3+ Treg cells are reduced in number. Furthermore, CD25 expression is marginally reduced among Foxp3+ Treg cells in conditional MHC-II-deficient mice, indicative of a lack of MHC-II-dependent TCR stimulation and/or IL-2 availability, and IL-2 administration in vivo caused greatly increased cell division among adoptively transferred Treg cells. This is not to say that IL-2 can cause Treg cell division in the complete absence of MHC-II as small numbers of MHC-II-bearing cells do remain in conditional MHC-II-deficient mice. Rather, this suggests only that IL-2 was limiting. Thus, our findings lend support to the proposal that Treg cell homeostasis depends on a delicate balance with a population of self-reactive IL-2-producing CD4+CD25+ cells which are themselves at least in part MHC-II-dependent.

We investigated the role of B cell Ag presentation in homeostasis of the memory B cell compartment in a mouse model where a conditional allele for the β-chain of MHC class II (MHC-II) is deleted in the vast majority of all B cells by cd19 promoter-mediated expression of Cre recombinase (IA-B mice). Upon T cell-dependent immunization, a small number of MHC-II+ B cells in IA-B mice dramatically expanded and restored normal albeit delayed levels of germinal center (GC) B cells with an affinity-enhancing somatic mutation to Ag. IA-B mice also established normal levels of MHC-II+ memory B cells, which, however, subsequently lost MHC-II expression by ongoing deletion of the conditional iab allele without significant loss in their number. Furthermore, in vivo Ag restimulation of MHC-II− memory B cells of IA-B mice failed to cause differentiation into plasma cells (PCs), even in the presence of Ag-specific CD4+ T cells. In addition, both numbers and Ag-specific affinity of long-lived PCs during the late post-GC phase, as well as post-GC serum affinity maturation, were significantly reduced in IA-B mice. These results support a notion that MHC-II-dependent T cell help during post-GC phase is not absolutely required for the maintenance of memory B cell frequency but is important for their differentiation into PCs and for the establishment of the long-lived PC compartment.

Ponds in the Saijo Basin of western Japan contain three Nuphar taxa, two of which are threatened. It has been proposed previously that the rarest of the three, hitherto Nuphar japonica var. saijoensis, may have originated from natural hybridization between N. japonicaand Nuphar pumila subsp. oguraensis. To test this hypothesis, we conducted morphological analyses, pollen and seed fertility tests, and a RAPD analysis of all three pond-lilies. Individuals of the putative hybrid exhibit intermediate morphology, reduced pollen and seed viability, and genetic additivity in comparison to the other species. The putative hybrid is also limited geographically to an area of parental sympatry. Our findings support the hybrid origin of N. japonica var. saijoensis, which we recognize nomenclaturally as Nuphar × saijoensis. Loss and degradation of habitat due to urbanization is a major threat to the survival of this taxon. © 2002 Elsevier Science B.V. All rights reserved.