Modulation of SOCS protein expression influences the interferon responsiveness of human melanoma cells

<p>Abstract</p> <p>Background</p> <p>Endogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells.</p> <p>Methods</p> <p>Basal and interferon-alpha (IFN-α) or interferon-gamma (IFN-γ)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells. Over-expression of SOCS1 and SOCS3 proteins in melanoma cells was achieved using the PINCO retroviral vector, while siRNA were used to inhibit SOCS1 and SOCS3 expression. Tyr⁷⁰¹-phosphorylated STAT1 (P-STAT1) was measured by intracellular flow cytometry and IFN-stimulated gene expression was measured by Real Time PCR.</p> <p>Results</p> <p>SOCS1 and SOCS3 proteins were expressed at basal levels in melanocytes and in all melanoma cell lines examined. Expression of the SOCS1 and SOCS3 proteins was also enhanced following stimulation of a subset of cell lines with IFN-α or IFN-γ. Over-expression of SOCS proteins in melanoma cell lines led to significant inhibition of Tyr⁷⁰¹-phosphorylated STAT1 (P-STAT1) and gene expression following stimulation with IFN-α (IFIT2, OAS-1, ISG-15) or IFN-γ (IRF1). Conversely, siRNA inhibition of SOCS1 and SOCS3 expression in melanoma cells enhanced their responsiveness to interferon stimulation.</p> <p>Conclusions</p> <p>These data demonstrate that SOCS proteins are expressed in human melanoma cell lines and their modulation can influence the responsiveness of melanoma cells to IFN-α and IFN-γ.</p

Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers

Alloprimed antibody-suppressor CD8+ T cells preferentially kill alloprimed germinal center B cells

Abstract Antibody-mediated rejection is a significant contributor to transplant rejection. Our prior work identified a novel subset of CXCR5+CD8+ T cells (CD8+ TAb-supp cells) that suppresses alloantibody production by mediating cytotoxic killing of IgG+ alloantibody-producing B cells. The purpose of these studies was to investigate which B cell subsets are susceptible to CD8+ TAb-supp cell-mediated cytotoxicity. Alloprimed CXCR5+CD8+ T cells were isolated from splenocytes of C57BL/6 mice on day 7 following allo-lysate stimulation. Various target cells (alloprimed B cell subsets, macrophages, and dendritic cells) were isolated from CD8 KO mice on day 7 following allo-lysate stimulation. Alloprimed CD8 KO mice are known to be high alloantibody producers from our prior studies. Target cells were tested in co-cultures to evaluate their susceptibility to CXCR5+CD8+ T cell-mediated in vitro cytotoxicity. Alloprimed CXCR5+CD8+ T cells exhibited significantly higher in vitro cytotoxicity towards alloprimed GC B cells (IgG+GL-7+Fas+; 11.4±1.0%) than towards other antibody producing B cell subsets, including alloprimed extrafollicular plasmablasts (IgG+CD138+; 0.9±0.3%) and plasma cells (IgG−CD138+; 1.6±0.5%; p&amp;lt;0.0001 for both). In addition, CXCR5+CD8+ T cells did not kill alloprimed dendritic cells (MHCII+CD11c+; 1.5±0.7) or macrophages (F4/80+; 1.7±0.4) in co-culture. No significant cytotoxicity was observed in control co-cultures using third party-primed CXCR5+CD8+ T cells (1.1±0.2), naïve CD8+ T cells (1.2±0.3%), or alloprimed CXCR5-negative CD8+ T cells (0.6±0.3%). These data show that alloprimed antibody-suppressor CXCR5+CD8+ T cells preferentially mediate cytotoxic killing of alloprimed GC B cells. This work was supported by a National Institutes of Health R01 grant AI083456 (to GLB), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.</jats:p

Phenotypic Distribution of Nerodia erythrogaster in Extreme Southeastern Illinois, Western Kentucky, and Adjacent Western Tennessee, USA

Volume: 67Start Page: 109End Page: 11

Surface uplift above the Jemez mantle anomaly in the past 4 Ma based on 40Ar/39Ar dated paleoprofiles of the Rio San Jose, New Mexico, USA

We combine 15 new 40Ar/39Ar ages with existing age constraints of basalts to investigate the incision and denudation history of the ~150-km-long Rio San Jose (RSJ) of west-central New Mexico (USA) over the past 4 Ma. Temporal and spatial scales of differential incision may help evaluate the relative importance of neotectonic, geomorphic and climatic forcings. The RSJ is a southeast-flowing river that orthogonally crosses the northeast-trending Jemez volcanic lineament, which is underlain by a zone of low-velocity mantle. Preserved basalt flows along the length of the river at different elevations that directly overlie river gravels are used to construct paleoprofiles of the RSJ and give insight into the differential incision history, which can test the hypothesis that epeirogenic uplift associated with the Jemez lineament influenced differential incision of the RSJ. Observations include (1) a northeast-trending graben along the central reach of the RSJ (El Malpais valley graben) which is parallel to the Jemez lineament, (2) the present-day east tilt of the originally west-flowing 3.7 Ma Mesa Lucero flow along the eastern edge of the Jemez lineament, and (3) modern profile convexities that are colocated with ca. 3 Ma paleoprofile convexities and are centered above the Jemez lineament. The arched ca. 3 Ma paleoprofile defined by the pre–Mount Taylor strath has greater convexity than younger profiles, suggesting neotectonic bowing of ~135 m (~50 m/Ma) in this reach over the past ~3 Ma relative to areas off axis of the Jemez lineament, in spite of graben subsidence and aggradational fill in this reach exceeding 100 m. Differential incision of the 184 ka Suwanee flow at the edge of the Colorado Plateau may be attributable to base-level fall in downstream reaches of the RSJ and/or headwater uplift, and more erosive climate in the past several hundred thousand years. However, these observations, when considered together, cannot be explained entirely by geomorphic or climatic forcings. Rather, they are best interpreted as resulting from surface uplift centered over the northeast-trending Jemez lineament, and our model suggests that both the faulting and broad bending may relate to mantle driven epeirogeny that caused differential river incision. Several interacting neotectonic and magmatic mechanisms may have contributed to postulated uplift. Magmatically driven geodynamic uplift forcings may include construction of the Mount Taylor stratovolcano just north of the RSJ that changed surface elevation by several kilometers at the volcanic peak itself. However, semisteady denudation and similar incision rates in other rivers in the region indicate that a regional erosional landscape was the primary driver of differential river incision over the past 5–8 Ma. Our focus on the pre–Mount Tayler RSJ paleoprofile reinforces this conclusion. Other mantle-related uplift mechanisms that may have generated mantle buoyancy include thermal buoyancy or magmatic inflation due to dike and sill networks related to the building of the Mount Taylor stratovolcano and eruption of Zuni-Bandera volcanic fields. Both could have contributed to uplift, but their relative importance is unknown. Broad epeirogenic uplift is also possible due to small-scale upper mantle convection beneath a thin elastic plate and resulting dynamic topography

Deficiency of antibody-suppressor CXCR5+CD8+ T cells (not CD4+ Tregs) drives high alloantibody production in CCR5 KO kidney transplant recipients

Abstract Kidney transplant (KTx) into CCR5 KO mice is an excellent model to study AMR immunobiology due to a heightened humoral response posited to be secondary to impaired CD4+Treg cell trafficking to the allograft. We have observed that adoptive cell therapy (ACT) with alloprimed CXCR5+CD8+ T cells mediates significant reduction of alloAb titer in CCR5 KO KTx recipients. We hypothesized that CXCR5+CD8+ T cells are more potent inhibitors of alloAb production than CD4+Tregs. We found that CCR5 KO recipients have significantly fewer CXCR5+CD8+ T cells (782±183 vs 2058±167 cells/mm3) and graft-infiltrating CD4+Treg cells (CD25+FoxP3+; 12.9±8.8 vs 56.2±8.8 cells/mm3) compared to WT recipients, as well as significantly greater quantity of splenic germinal center (GC; GL7+Fas+B220+) B cells (5814±436 vs 671±342 cells/mm3; all p&amp;lt;0.003). CCR5 KO recipient alloAb titer is significantly reduced following CXCR5+CD8+ T cell ACT (1259±436 vs 5951±404 in untreated controls; p&amp;lt;0.0001) but not following CD4+Treg ACT (5688±436). CXCR5+CD8+ T cells reduced splenic GC B cell (5813±589 to 4087±537; p=0.002) quantity, while CD4+Treg had no impact (6916±538/mm3). Compared to untreated controls, CD8-depletion in WT recipients led to significant increase in alloAb titer (1508±338 vs 6078±436) and GC B cells (671±436 vs 7761±430; p&amp;lt;0.0001 for both). CD4+Treg depletion led to increased alloAb titer (3180±436) and GC B cell quantity (4911±429; p&amp;lt;0.004 for both), though less pronounced than in the CD8-depleted group (both p&amp;lt;0.001). This suggests that CXCR5+CD8+ T cells are more potent regulators of alloAb production than CD4+Tregs and their deficiency in CCR5 KO recipients is the major driver of heightened humoral immunity and AMR following KTx. Supported by NIH R01 AI083456 (to GLB), T32 AI106704-07 and F32 AI161844 (to JLH), CA016058, UL1TR002733, the OSU Division of Transplant Surgery, and the OSU College of Medicine.</jats:p

Distinct phenotype of antibody-suppressor CXCR5+CD8+ T cells

Abstract The novel CXCR5+CD8+ T cell subset has been shown to inhibit alloantibody production (termed CD8+ TAb-supp cells) but do not reject allografts. In contrast, CXCR3+CD8+ T cells reject allogeneic transplants (but do not inhibit antibody). Recent literature indicates there are many CXCR5+CD8+ T cell subsets with different effector functions (anti-viral, anti-tumor, anti-autoimmune, or antibody enhancement). By investigating CD8+ TAb-supp cell by RNA-seq and flow cytometry, we aimed to determine a profile that distinguishes this novel subset. To investigate this, C57BL/6 (H-2b) were transplanted with FVB/N (H-2q) hepatocytes and CD8+ T cell subsets were analyzed on day 7. Alloprimed CXCR5+CD8+ T cells were compared to naïve CD8+ T cells and alloprimed CXCR3+CD8+ T cells. Fluorescence minus one controls were used to determine background staining (flow cytometry). RNA-seq analysis show that 1670 transcripts were upregulated or downregulated when comparing between flow-sorted alloprimed (CD62L−CD44+) CXCR5+CD8+ T cells or naïve (CD62L+CD44−) CD8+ T cells. Transcripts of note include Bcl-6 (3.3-fold upregulated), CXCR3 (8.4-fold downregulated), and S1PR3 (140-fold upregulated). Flow cytometry analysis suggests that CD8+ TAb-supp cells are short-lived effectors cells based on the expression of CD44, KLRG1, and CD122 (not CD62L or CD127). An expansive flow analysis panel comparing differences in transcript and protein expression between alloprimed CXCR5+CD8+ TAb-supp cells and other reported CXCR5+CD8+ T cell subsets indicates that the absence of PD-1, IL-10, and FoxP3 expression distinguishes CD8+ TAb-supp cells from all reported CXCR5+CD8+ T cell subsets (including CD8+ T follicular regulatory cells).</jats:p