Non-lymphoid Cells Research Articles

Lymphocyte Phosphatase-Associated Phosphoprotein Is a Substrate of Protein Kinase CK2.

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a molecular partner of CD45 phosphatase that plays a key role in the regulation of antigen-specific activation of lymphocytes. The functions of LPAP still remain unknown. We believe that studying LPAP phosphorylation pathways could shed light on its functions. In this work, we studied the phosphorylation of LPAP ectopically expressed in non-lymphoid cells in order to determine the effect of LPAP interaction partners on its phosphorylation. We found that phosphorylation at Ser153 and Ser163 in non-hematopoietic HEK293 cells was conserved, while phosphorylation at Ser99 and Ser172 was almost absent. The pattern of LPAP phosphorylation in K562 erythroid and U937 myeloid cells expressing endogenous CD45 protein was similar to that observed in T and B lymphocytes. We demonstrated for the first time that LPAP is a substrate for protein kinase CK2 that phosphorylates it at Ser153, presumably ensuring LPAP resistance to degradation.

CD8+ T cells promote cytokine responses to stress

Psychological stress is known to have profound effects on immune function and to promote inflammatory conditions. Elevated circulating levels of cytokines associated with stress are known to increase the risk to several diseases, but little is known about this mechanism. This study assessed the role of T cells on cytokine levels after exposure to stress in the learned helplessness paradigm. Adoptive transfer of CD4+ T cells into Rag2−/− mice did not change cytokine levels to stress while CD8+ T cells resulted in an increase in TNF-α, IL-6 and IFN-γ in stressed Rag2−/− mice. Moreover, depletion of CD8+ T cells in WT mice abolished these cytokine responses to stress. Corticosterone and behavioral stress responsiveness was impaired in Rag2−/− mice reconstituted with CD8+ T cells. Notably, depletion of these cells in WT mice had no effect on behavior or corticosterone levels. Exposure to stress did not change the expression of canonical markers of T cell activation including CD62L and CD44 or modified intracellular cytokine content, suggesting that they are not the main producers of circulating cytokines in response to stress. These results show that CD8+ T cells promote TNF-α, IL-6 and IFN-γ responses to stress, possibly by stimulating non-lymphoid cells.

Abstract 3173: Lymphocyte-specific chromatin accessibility predetermines glucocorticoid resistance in acute lymphoblastic leukemia

Abstract Glucocorticoids are well known for their immunosuppressive activity, and play a critical role in the treatment of lymphoid malignancies. However, the development of resistance remains a significant barrier to cure and the mechanisms are poorly defined. Moreover, glucocorticoids are rarely efficacious in treating myeloid and other non-lymphoid malignancies, and the mechanisms of lymphocyte-specific efficacy are unclear. To address these issues, we first carried out a global analysis of DNase I hypersensitive sites in 18 lymphoid and 64 non-lymphoid cell types to map lymphocyte-specific open chromatin domains (LSOs). We then integrated these domains with genome-wide glucocorticoid-induced gene transcription and epigenetic modulation in an in vivo patient-derived xenograft (PDX) model of acute lymphoblastic leukemia (ALL). Performing chromatin immunoprecipitation and assays for transposase-accessible chromatin (ATAC), we determined a strong correlation between glucocorticoid receptor (GR) binding and chromatin accessibility, acetylated histone marks and binding of a DNA structural protein (CTCF), and identified 1,536 GR bound LSOs. We next analyzed RNA-seq data in glucocorticoid sensitive and resistant ALL PDXs for expression changes in genes located within 100 kb of the LSOs after glucocorticoid treatment in vivo, and identified four groups comprising 389 genes that were significantly differentially expressed. Of the 198 up-regulated genes, 143 showed increased H3K27Ac enrichments at 177 LSOs. Forty-two LSOs showed the increase only in glucocorticoid sensitive but not resistant ALLs. Applying this to an extended panel of ALL PDXs, basal DNA methylation at the 42 LSOs was significantly higher in resistant ALLs despite no difference within the gene bodies, whereas the basal chromatin accessibility indicated by ATAC abundance was diminished. Pathway analysis indicated that the LSO regulated genes were involved in repressing B- and T- cell receptor signaling pathways and activating the apoptotic pathway. One such LSO was at the pro-apoptotic BIM gene locus, where CTCF binding was found only in lymphocytes but not in other cell types. The GR cooperated with CTCF to mediate interactions between the promoter and the BIM LSO to direct DNA looping, thus triggering BIM transcription. Importantly, this LSO was heavily methylated in resistant ALLs as well as non-lymphoid cells. Azacitidine, a DNA demethylating drug that is routinely used in the clinic, could partially reverse these changes and restore glucocorticoid sensitivity. Taken together, this study demonstrated for the first time that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell type. Reversal of these epigenetic changes may lead to improvements in the use of glucocorticoids in the clinic. Citation Format: Duohui Jing, Yizhou Huang, Xiaoyun Liu, Keith Sia, Rebecca C. Poulos, Miriam Span, Chao Zhang, Jianqing Mi, Jason WH Wong, Dominik Beck, John E. Pimanda, Richard B. Lock. Lymphocyte-specific chromatin accessibility predetermines glucocorticoid resistance in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3173.

The t(14;18) translocation is absent from endothelial and follicular dendritic cells of follicular lymphoma (FL) and shows heterogeneous presence in preserved FL mantle zones

BackgroundThe translocation t(14;18)(q32;q21) is the genetic hallmark of follicular lymphoma (FL) and can be observed in 85–90% of cases. Whether the translocation is restricted to cells with germinal center B-cell phenotype or can be observed in other cell types of the microenvironment remains debated. Of interest, cases of associated histiocytic and dendritic cell sarcomas arising in the background of FL have been shown to be clonally related and carry the t(14;18), suggesting a “transdifferentiation” of the malignant FL clone into a neoplasm of a different hematopoietic lineage.MethodsWe analyzed the presence of the t(14;18)(q32;q21) as a surrogate marker of the malignant clone in cells of the FL microenvironment using combined fluorescence immunophenotyping and interphase cytogenetics targeting the BCL2 gene locus. In addition to non-lymphoid cells in FL, we analysed FL with preserved IgD+ mantle zones and cases of in situ follicular neoplasia (ISFN) to investigate whether cells of non-germinal center B-cell phenotype are part of the malignant clone.ResultsSix (40%) of 15 manifest FL cases with preserved IgD+ mantle zones did not harbour the t(14;18)(q32;q21) translocation. In all t(14;18) + FL cases, follicular dendritic cells and endothelial cells lacked the t(14;18) translocation. 2/9 FL revealed t(14;18)- IgD+ mantle zone B-cells. In the seven ISFN cases, the t(14;18) translocation was strictly confined to germinal center cells.ConclusionsThe t(14;18) translocation in follicular lymphoma is limited to B-cells. The origin of IgD+ mantle cells is heterogeneous, in the majority of cases belonging to the neoplastic clone, whereas a minority of cases of manifest FL show nonneoplastic mantle zones, similar to ISFN.

Atrophied thymus can serve as a tumor reservoir for harboring melanoma cells

Abstract Tumor metastatic relapse is responsible for main cancer associated mortality and potentially arises from undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy at a dormant state hiding in certain organs (termed: tumor reservoirs). A central T cell immune organ, the thymus, has been suggested as this kind of pre-metastatic tumor reservoir for B-lymphoma cells, which are a risk for eventual relapse. It remains unknown whether the thymus is able to harbor non-lymphoid solid tumor cells, why chemotherapy cannot thoroughly eliminate cancer cells in the thymus, and what the state of thymic occult cancer cells is during chemotherapy. With melanoma inoculated and genotoxic doxorubicin treated mouse model, we determined that the thymus, particularly the atrophied thymus, was able to harbor blood stream-circulating melanoma cells. We found that chemotherapy-resulted DNA-damage response triggered p53 activation in non-malignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. This inflammatory condition induced thymic occult minimal tumor cells to acquire heterogenic chemo-resistant dormancy. Therefore, the thymus, which becomes a pre-metastatic reservoir for non-lymphoid solid tumor cells under chemotherapy, should be a novel target in antitumor therapy for considering and preventing from tumor metastatic relapse.

Regulation and In Vivo Function of Dendritic Cell CTLA-4

Abstract CTLA-4, the target of the blockbuster cancer immunotherapeutic ipilimumab, is the best characterized of the T-cell immune checkpoints. In contrast to T-cell expressed CTLA-4, almost nothing is known about CTLA-4 expression or function in non-lymphoid cell types. Recently we reported expression and secretion of microvesicle-bound CTLA-4 from myeloid lineage dendritic cells. Previous data indicated that microvesicular CTLA-4 bound B7, leading to vesicle internalization and downregulation of B7 expression in vitro. Further, siRNA knockdown of DC-expressed CTLA-4 resulted in upregulation of CD8+ cell proliferation and enhanced antitumor and antiviral immunity in vitro and in vivo. Here we report that DC CTLA-4 can be regulated by interferons a, g, and especially b, whereas TLR ligands appeared to have little effect on regulation. Additional experiments suggested negative regulation of DC CTLA-4 by miR-223 and the transcription factor C/EBP-b. To better understand the role of CTLA-4 in dendritic cell function in vivo, Balb/c conditional knockout animals were generated and characterized under conditions of homeostasis and following nonlethal challenge with H3N2 A/HongKong/8/68 influenzavirus. T-cells derived from conditional knockout animals lacked expression of CD62L and exhibited downregulated CCR7 expression with upregulation of exhaustion markers PD-1 and GITR. Knockout animals challenged with influenza exhibited higher levels of NP tetramer-specific CD8+CD25+ cells and a lengthily extended period of morbidity. The sum total of the data reflect the broad hypothesis that dendritic cell CTLA-4 serves as a regulatory checkpoint to prevent non-specific, inappropriate, or excessively pathologic T-cell priming.

Immune regulation by Tim-3.

T-cell immunoglobulin and mucin domain 3 (Tim-3) is a transmembrane protein that in both mice and humans has been shown to possess various functions in a context-dependent manner. Thus, Tim-3 has been associated with both inhibitory and co-stimulatory function, depending in part on the specific cell type and immune response course. Though originally described on T cells, Tim-3 is now known to be expressed by both lymphoid and non-lymphoid cells within the immune system and even by non-immune cells. In addition, though widely thought of as a negative regulator of immunity, Tim-3 has been shown in more recent studies to have a positive function on both myeloid and lymphoid cells, including T cells. Tim-3 is often expressed at a high level on exhausted T cells in tumors and chronic infection and may engage in crosstalk with other so-called “checkpoint” molecules such as PD-1. Thus, Tim-3 has emerged as a possible therapeutic target, which is being actively explored both pre-clinically and clinically. However, recent research suggests a more complex in vivo role for this protein, compared with other targets in this area.

Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci.

CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors.

T Cells in Nonlymphoid Tissues Give Rise to Lymph-Node-Resident Memory T Cells

Immunosurveillance of secondary lymphoid organs (SLO) is performed by central memory Tcells that recirculate through blood. Resident memory T (Trm) cells remain parked in nonlymphoid tissues and often stably express CD69. We recently identified Trm cells within SLO, but the origin and phenotype of these cells remains unclear. Using parabiosis of "dirty" mice, we found that CD69 expression is insufficient to infer stable residence of SLO Trm cells. Restimulation of nonlymphoid memory CD8+ Tcells within the skin or mucosa resulted in a substantial increase in bona fide Trm cells specifically within draining lymph nodes. SLO Trm cells derived from emigrants from nonlymphoid tissues and shared some transcriptional and phenotypic signatures associated with nonlymphoid Trm cells. These data indicate that nonlymphoid cells can give rise to SLO Trm cells and suggest vaccination strategies by which memory CD8+ Tcell immunosurveillance can be regionalized to specific lymph nodes.

Unravelling the roles of innate lymphoid cells in cerebral malaria pathogenesis.

Cerebral malaria (CM) is one complication of Plasmodium parasite infection that can lead to strong inflammatory immune responses in the central nervous system (CNS), accompanied by lung inflammation and anaemia. Here, we focus on the role of the innate immune response in experimental cerebral malaria (ECM) caused by blood-stage murine Plasmodium berghei ANKA infection. While T cells are important for ECM pathogenesis, the role of innate lymphoid cells (ILCs) is only emerging. The role of ILCs and non-lymphoid cells, such as neutrophils and platelets, contributing to the host immune response and leading to ECM and human cerebral malaria (HCM) is reviewed.

Presence and Levels of Anti-GM3 Antibodies and GM3 Ganglioside Molecules in Probes from Experimental In Vitro-Models

Background: The current study was directed to determine the values of both GM3 ganglioside and specific anti-GM3 antibodies in in vitro-incubated normal cells, malignant cell and mixed cultures of co-cultivated cells from both types as well. So a better understanding of the molecular mechanisms, underlining differences between various cellular types and changes during the process of cell differentiation was necessary. Methods: Total lysates from cultivated normal cells from mouse embryos, mouse malignant myeloma line and mixed of both were prepared and passed through GSHAgarose Columns. Molecules with affinity to the reduced form of Glutathione (GSH) were separated. The levels of ganglioside GM3 and of specific antibodies to it were assessed by enzyme-linked immuno-sorbent assay (ELISA) technique. Results: In the normal cells’ lysate, supplemented with molecules, possessing affinity to GSH the lowest values were assessed. These differences could be due to contention of many non-possessing such affinity molecules in equal volume of biological material, as well as with the presence of various bonded forms of GM3. Conclusions: The data obtained confirm literature data about the increased levels of GM3 as a marker for malignancy. We propose derivation of lymphoid-like cells from the embryonic progenitors. Another hypothesis could be the production of immunoglobulins (IgG antibodies) from non-lymphoid cells in appropriate conditions.

Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry.

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+ dependent and the other is Ca2+ independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Lymph node cytology in the dog and cat

Lymph node (LN) cytology, a cost-effective and highly rewarding diagnostic technique, is performed routinely in the everyday practice. Localised or generalised lymphadenomegaly and clinical staging of malignant tumors comprise the main indications of this valuable diagnostic modality. The superficial and easily palpated submandibular, prescapular and popliteal LN can be easily positioned, while internal LN (i.e. mediastinal, mesenteric, sublumbar) may necessitate a surgical exploration or ultrasound guidance. Fine-needle biopsy and imprint smears from excised LN are the most common techniques to obtain the necessary material. Small lymphocytes constitute more than 80% of the normal LN population, with medium and large lymphocytes making up approximately the 5-15% of it. The occasional presence of plasma and mast cells, neutrophils, eosinophils and macrophages can also be noticed. Lymphadenomegaly, either localised or generalised, is mainly due to hyperplasia, inflammation or neoplasia. Hyperplastic LN may appear normal cytologically, but typically, small lymphocytes predominate (>50% of nucleated cells [NC]), in the context of a moderate increase of medium/large lymphocytes (<20%of NC) and plasma cells (>5% of NC). Hyperplastic lymphadenopathy is indicative of a lymphoid proliferation secondary to a local (e.g. deep staphylococcal pyoderma) or generalised (e.g. leishmaniosis) antigenic stimulation. In primary or secondary lymphadenitis, LN are infiltrated by increased numbers of inflammatory cells, the predominance of which define the neutrophilic or purulent, eosinophilic, granulomatous or pyogranulomatous inflammation. Bacterial (e.g. Mycobacterium sp.), fungal (e.g. Histoplasma capsulatum) and protozoal (e.g. Leishmania infantum) infections, represent the leading etiologies of canine and feline lymphadenitis. In lymphoma, the most common neoplastic lymphadenopathy in both animal species, the predominance of medium and large (> 50% of NC) immature lymphocytes is observed. Metastatic neoplasia can be documented when non-lymphoid malignant cells (i.e. epithelial, mesenchymal) or increased numbers of normally present cells (i.e. mast cells), also bearing malignant features, are detected on LN cytology. Failure to establish a definitive diagnosis on LN cytology warrants a surgical biopsy and histopathology.

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

Cyclin D1 expression by histiocytes may mimic cyclin D1-positive proliferation centres of chronic lymphocytic leukaemia/small lymphocytic lymphoma

AimsCyclin D1, generally considered to be absent in chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), has been reported in the proliferation centres (PCs) of recent CLL/SLL cases. Cyclin D1 immunostaining in CLL/SLL may lead to diagnostic confusion. The objective of this study was to identify the types of stained cells and the impact on diagnosis. MethodsCyclin D1 expression was assessed by immunostaining samples from 46 cases of CLL/SLL. CD68 and double immunostaining with CD20/CyclinD1, CD68/CyclinD1, and CD163/CyclinD1 were then performed in cases of CLL/SLL positive for cyclinD1 in the PCs. ResultsDim-positive cyclin D1 staining in randomly scattered cells in the CLL/SLLs were observed in 38/46 cases (82.6%). In five (10.9%) cases, more than 50 cyclin D1-positive cells per high-power field were detected within the PCs in CLL/SLL with weak to moderate intensity. Double immunochemical staining in these cases showed that cyclin D1 in these positive cells was mostly co-expressed with CD68 and CD163 and the cells were negative for CD20. ConclusionsThe cyclin D1-positive CLL/SLL cells in this study were mostly histiocytes. The expression of cyclin D1 by histiocytes may mimic cyclin D1+ CLL/SLL; thus, the recognition of cyclin D1 expression by non-lymphoid cells in lymphoma is important.

INCB040093 Is a Novel PI3Kδ Inhibitor for the Treatment of B Cell Lymphoid Malignancies.

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin's lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1β (MIP-1beta) and tumor necrosis factor-β (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin's lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

Interleukin-18: a regulator of cancer and autoimmune diseases.

Interleukin (IL)-18, structurally similar to IL-1β, is a member of IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and nonlymphoid cells, has an important role in inflammatory processes. The main function of IL-18 is mediated through induction of interferon-γ (IFN-γ) secretion from T helper (Th1) cells. This cytokine synergistically with IL-12 contributes to Th1 differentiation and, therefore, is important in host defense mechanisms against intracellular bacteria, viruses, and fungi. Recent evidences showing the involvement of IL-18 in Th2 differentiation and ultimately IgE production from B cells have shed a new insight on the dual effects of IL-18 on Th1 and Th2 inflammatory responses. IL-18 in combination with IL-12 can activate cytotoxic T cells (CTLs), as well as natural killer (NK) cells, to produce IFN-γ and, therefore, may contribute to tumor immunity. The biological activity of IL-18 is not limited to these cells, but it also plays a role in development of Th17 cell responses. IL-18 synergistically with IL-23 can induce IL-17 secretion from Th17 cells. The diverse biological activity of IL-18 on T-cell subsets and other immune cells has made this cytokine a good target for investigating its role in various inflammatory-based diseases. Lately, the discovery of IL-18 binding protein (IL-18BP), a physiological inhibitor of IL-18 and a hallmark of IL-18 biology, made this cytokine an attractive target for studying its pros and cons in the treatment of various diseases. In recent years, the biology, genetics, and pathological role of IL-18 have been studied in a number of diseases. In this article, we aimed to present an updated review on these aspects regarding the contribution of IL-18 to important diseases such as cancer, autoimmunity, and inflammatory-mediated conditions including allergic diseases, metabolic syndrome, and atherosclerosis. Emerging data indicating prognostic, diagnostic, and therapeutic features of IL-18 and its related molecules will also be discussed.

Extrinsically derived TNF is primarily responsible for limiting antiviral CD8+ T cell response magnitude.

TNF is a pro-inflammatory cytokine produced by both lymphoid and non-lymphoid cells. As a consequence of the widespread expression of its receptors (TNFR1 and 2), TNF plays a role in many important biological processes. In the context of influenza A virus (IAV) infection, TNF has variably been implicated in mediating immunopathology as well as suppression of the immune response. Although a number of cell types are able to produce TNF, the ability of CD8+ T cells to produce TNF following viral infection is a hallmark of their effector function. As such, the regulation and role of CD8+ T cell-derived TNF following viral infection is of great interest. Here, we show that the biphasic production of TNF by CD8+ T cells following in vitro stimulation corresponds to distinct patterns of epigenetic modifications. Further, we show that a global loss of TNF during IAV infection results in an augmentation of the peripheral virus-specific CD8+ T cell response. Subsequent adoptive transfer experiments demonstrated that this attenuation of the CD8+ T cell response was largely, but not exclusively, conferred by extrinsic TNF, with intrinsically-derived TNF making only modest contributions. In conclusion, TNF exerts an immunoregulatory role on CD8+ T cell responses following IAV infection, an effect that is largely mediated by extrinsically-derived TNF.

HTLV-1-infected thymic epithelial cells convey the virus to CD4+ T lymphocytes

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+T cells are the main target of HTLV-1, but other cell types are known to be infected, including immature lymphocytes. Developing T cells undergo differentiation in the thymus, through migration and interaction with the thymic microenvironment, in particular with thymic epithelial cells (TEC) the major component of this three dimensional meshwork of non-lymphoid cells. Herein, we show that TEC express the receptors for HTLV-1 and can be infected by this virus through cell–cell contact and by cell-free virus suspensions. The expression of anti-apoptosis, chemokine and adhesion molecules genes are altered in HTLV-1-infected TEC, although gene expression of antigen presentation molecules remained unchanged. Furthermore, HTLV-1-infected TEC transmitted the virus to a CD4+ T cell line and to CD4+ T cells from healthy donors, during in vitro cellular co-cultures. Altogether, our data point to the possibility that the human thymic epithelial cells play a role in the establishment and progression of HTLV-1 infection, functioning as a reservoir and transmitting the virus to maturing CD4+ T lymphocytes, which in turn will cause disease in the periphery.

EFhd2/ Swiprosin-1 regulates the cytoskeleton of B lymphocytes and impacts germinal center reaction

Abstract The germinal center (GC) reaction is a decisive checkpoint for efficient humoral immunity. Most GC associated processes, e.g. cell migration and cell adhesion, are strongly dependent on cytoskeletal dynamics and B cell receptor (BCR) signaling. However, the molecular connection between downstream BCR molecules and cytoskeletal proteins is not yet fully elucidated. The adaptor protein EFhd2/ Swiprosin-1 co-localizes with the BCR at the plasma membrane. It controls F-Actin bundling as well as Actin remodeling through Cofilin activity and is involved in spreading, lamellipodia formation and migration in non-lymphoid cells. We showed that constitutively EFhd2 deficient mice display enhanced GC responses upon challenge with the helminth Nippostrongylus brasiliensis in a B cell intrinsic manner in mixed bone marrow chimeras. Moreover, B cell specific deletion of EFhd2 leads to enhanced GC B cell numbers already in steady state. We therefore hypothesize that EFhd2 controls the GC reaction by regulating cytoskeletal processes. In vitro transwell migration assays and in vivo studies using two-photon microscopy revealed decreased directed chemotaxis but increased random chemokinesis of EFhd2−/− B cells. Moreover, activated B cells spread faster in the absence of EFhd2 on anti-BCR antibodies which correlates with accelerated Cofilin activity. Finally, also BCR capping was increased in activated EFhd2 deficient B cells. Taken together, we suggest that EFhd2 controls Actin dynamics through Cofilin in B cells, thereby, controlling chemotaxis of B cells and BCR mediated B cell spreading. Hence, EFhd2 links proper coordination of the B cell cytoskeleton with the GC checkpoint and impacts TH2 response.