IFN-α Is Not Sufficient to Drive Th1 Development Due to Lack of Stable T-bet Expression

Ramos, Hilario J.; Davis, Ann M.; George, Thaddeus C.; Farrar, J. David

doi:10.4049/jimmunol.179.6.3792

Cited by 38 publications

(36 citation statements)

References 62 publications

(66 reference statements)

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“…17 However, IFNAR is thought to be constitutively expressed on all cells, enabling them to respond in an autocrine fashion to IFN-␣ that is secreted during viral infections. 6 Thus, selective IFNAR expression has not been examined during T-cell differentiation.…”

Section: Reciprocal Regulation Of the Il-12r And Ifnar In T Em And T CMmentioning

confidence: 99%

“…Detection of tyrosinephosphorylated STAT1 and STAT4 was performed by intracellular staining, as described. 17 Cells were stained with unconjugated rabbit polyclonal antibodies to STAT1 (clone SC-346), STAT-4 (clone SC-486; Santa Cruz Biotechnology), or antiphosphotyrosine STAT1 (Upstate Biotechnology, Lake Placid, NY) or antiphosphotyrosine STAT4 (Zymed Laboratories, San Francisco, CA). A goat anti-rabbit Ig-biotin (Jackson ImmunoResearch Laboratories, West Grove, PA) was used for secondary detection, followed by streptavidin-PerCP (BD Biosciences).…”

Section: Intracellular Stat Stainingmentioning

confidence: 99%

See 1 more Smart Citation

Reciprocal responsiveness to interleukin-12 and interferon-α specifies human CD8+ effector versus central memory T-cell fates

Ramos¹,

Davis²,

Cole³

et al. 2009

Blood

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IntroductionCD8 ϩ T cells are critical mediators of adaptive inflammatory responses to intracellular pathogens. They require a series of signals for efficient expansion and acquisition of effector functions, such as cytokine secretion and lytic activity. These signals are delivered by professional antigen-presenting cells (APC) and include antigen recognition (signal 1), costimulatory activation (signal 2), and signaling provided by innate inflammatory cytokines (signal 3). 1 Whereas signals 1 and 2 prime naive CD8 ϩ T cells and initiate cell division, signal 3 cytokines program effector functions and ensure clonal survival. A variety of cytokines have the potential to act as signal 3 in CD4 ϩ and CD8 ϩ T cells, 2-4 and interleukin (IL)-12 and interferon (IFN)-␣/␤, in particular, promote efficient induction of innate immunity as well as the development of adaptive type 1 responses to intracellular infection. 5,6 Thus, IL-12 and IFN-␣/␤ may represent the predominant signal 3 during intracellular infection.Whereas IL-12 regulates T helper (Th) 1 development in CD4 ϩ T cells, early reports suggested that the induction of IFN-␥ secretion and lytic activity in CD8 ϩ T cells was independent of IL-12, signal transducer and activator of transcription (STAT) 4, and T-box expressed in T cells (T-bet). [7][8][9] However, recent studies by Schmidt and Mescher found that in vitro priming with IL-12 induced high and sustained secretion of IFN-␥ and markedly enhanced lytic activity in murine CD8 ϩ T cells. 10 Furthermore, these effects were dependent on STAT4, indicating that IL-12 signaling provides a necessary third signal for the regulation of CD8 ϩ T-cell development. 3,11,12 More recent studies have indicated that IFN-␣/␤ can act in a manner similar to IL-12 to provide signal 3 and promote the induction of cytokine secretion, cytolytic activity, and clonal expansion in murine CD8 ϩ T cells. 3,13 Collectively, these studies suggested that IL-12 and IFN-␣ can act as redundant signals to promote the development of effector responses in murine CD8 ϩ T cells.In addition to enhancing effector cell development, IFN-␣/␤ was implicated in the generation of memory CD8 ϩ T cells in vivo. In these studies, IFN-␣/␤ receptor (IFNAR)-deficient, T-cell receptor (TCR)-transgenic (P14) CD8 ϩ T cells failed to expand and generate memory populations in response to in vivo lymphocytic choriomeningitis virus infection despite their ability to proliferate efficiently in vitro. 13 Alternatively, IL-12 Ϫ/Ϫ mice displayed defective primary effector responses, whereas development of T central memory (T CM ) cells was markedly enhanced compared with wild type, indicating that IL-12 signaling suppresses T CM development. [14][15][16] Considering that IFN-␣/␤ has been implicated in effector and memory cell development, it is unclear how this signal regulates both events and whether any of these activities operate in human CD8 ϩ T cells. Furthermore, it is not clear how IL-12 and IFN-␣/␤ signals are integrated to balance effector and memory cell develo...

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Section: Reciprocal Regulation Of the Il-12r And Ifnar In T Em And T CMmentioning

confidence: 99%

Section: Intracellular Stat Stainingmentioning

confidence: 99%

Reciprocal responsiveness to interleukin-12 and interferon-α specifies human CD8+ effector versus central memory T-cell fates

Ramos¹,

Davis²,

Cole³

et al. 2009

Blood

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“…ImageStream analysis of cell and nuclear shape combined with immunophenotype has been used by other investigators to define cells produced during ES cell differentiation, cells bound by natural killer cells, and which cells are producing IFN-gamma (Kim et al, 2007;Neff-LaFord et al, 2007;Woll et al, 2008). There are a growing number of investigators using the ImageStream to quantify colocalization of proteins or the extent of a protein's localization within the nucleus (Beum et al, 2006;Liu et al, 2007;Matsuda et al, 2007;Ramos et al, 2007). The combination of standard fluorescent detectors of aspects of apoptosis with examination of nuclear morphology has also been used to facilitate a more complete analysis of the apoptotic process and allow late stage apoptosis to be distinguished from necrosis (George et al, 2004;Guzman et al, 2007).…”

mentioning

confidence: 99%

Multispectral imaging of hematopoietic cells: Where flow meets morphology

McGrath

Bushnell

Palis

2008

Journal of Immunological Methods

120

118

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Normal and abnormal blood cells are typically analyzed by either histologic or flow cytometric approaches. Histology allows morphological examination of complex visual traits but with relatively limited numbers of cells. Flow cytometry can quantify multiple fluorescent parameters on millions of cells, but lacks morphological or sub-cellular spatial detail. In this review we present how a new flow technology, the ImageStream (Amnis Corporation, Seattle, WA), blends morphology and flow cytometry and can be used to analyze cell populations in ways not possible by standard histology or flow cytometry alone. The ImageStream captures brightfield, darkfield and multiple fluorescent images of individual cells in flow. The images can then be analyzed for levels of fluorescence intensity in multiple ways (i.e. maximum, minimum, or mean) as well as the shape and size of the area of fluorescence. Combinatorial measurements can also be defined to compare levels and spatial associations for multiple fluorescent channels. We demonstrate an application of this technology to distinguish six stages of erythroid maturation which have been classically defined by morphological criteria, by measuring changes in Ter119 mean intensity and area, DNA (Draq5 stain) mean intensity and area, and RNA content (thiazole orange stain). Using this approach, we find that other characteristics of erythroid maturational, such as marker expression and nuclear offset, vary appropriately within the defined cell subsets. Finally, we show that additional measurements of cell characteristics not classically analyzed in cytometry, including surface unevenness and unusually high contrast in brightfield images combined with fluorescent markers allow complex discriminations of rare populations.

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“…Further, in human, both Eomes and T-bet are regulated by IL-12 in CD8 ϩ and CD4 ϩ T cells, respectively. 18,26,37 As such, it is unclear how the IL-12 signaling pathway regulates only one of the 2 transcription factors in each cell type and whether species differences can account for this regulation.In addition to Eomes, this analysis revealed 4 additional unique IL-12-induced intracellular regulators that were expressed in T EM in vivo. First, we identified both a dual-specificity phosphatase (DUSP5) and a MAP kinase (MAP3K8), both of which have been implicated in directly regulating effector T-cell activity.…”

mentioning

confidence: 99%

IL-12 selectively programs effector pathways that are stably expressed in human CD8+ effector memory T cells in vivo

Chowdhury¹,

Ramos²,

Davis³

et al. 2011

Blood

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IntroductionThe pool of circulating CD8 ϩ cytotoxic T lymphocytes (CTLs) is remarkably heterogeneous, consisting of antigen inexperienced naive cells as well as multiple populations of effector and memory subsets. 1 Various models have been proposed to explain the genesis of memory subsets, and a significant body of evidence suggests that long-lived memory cells are derived from early effector cells that survive contraction as antigen loads wane. 2 If so, then the diverse spectrum of memory cells that fall into either effector memory (T EM ) or central memory (T CM ) categories would also be predicted to be derived from a common pool of primary effectors. 3 Regardless of their origins, T EM and T CM differ substantially in their functional capabilities, and the extrinsic signals that regulate their development are complex and multifactorial. Among these factors, innate cytokines are key signals that regulate effector cell differentiation, 4 with IL-12 and type I interferon (IFN-␣/␤) playing important roles in both effector and memory cell development. 5 In humans, CD8 ϩ subsets have been distinguished based on their differential expression of various markers, including CD45RA, CD27, CD28, CCR7, CD62L, CD127, and CXCR3. [6][7][8][9] Based solely on the expression of CCR7 and CD45RA, early studies defined 4 main subsets: naive (CD45RA ϩ /CCR7 ϩ ), T CM (CD45RA Ϫ / CCR7 ϩ ), T EM (CD45RA Ϫ /CCR7 Ϫ ), and effector memory-RA (T EMRA , CD45RA ϩ /CCR7 Ϫ ). 10 Aside from their differential expression of CD45RA and other cell surface markers, T EM uniformly exhibit rapid effector functions in response to antigen activation. Further, T EM do not generally require CD28-mediated costimulation or innate cytokines to secrete proinflammatory cytokines or to lyse target cells. 11,12 Thus, if T EM cells are direct descendants of effector CTLs, then the maintenance of effector functions within T EM would suggest early programming during the priming phase of infection.In mice, both IL-12 and IFN-␣/␤ exert somewhat overlapping and redundant effects by driving effector cell development. 5,[13][14][15][16][17] Although IL-12 seems to be more potent at promoting IFN-␥ secretion, both IL-12 and IFN-␣/␤ are equally effective at regulating lytic activity in murine CD8 ϩ T cells. In contrast, IL-12 and IFN-␣/␤ appear to play very distinct roles in priming human CD8 ϩ T-cell responses. Recently, Ramos et al demonstrated a preferential role for IL-12 over IFN-␣/␤ in driving cytokine expression and lytic activity in human CTLs. 18 Conversely, IFN-␣/␤ enhanced the development of a subpopulation of cells that displayed phenotypic and functional characteristics of T CM . Programming of effectors by IL-12 was accompanied by progression of cells through cell division and was altered by the strength of TCR engagement. IL-12 programmed effector cell development, which was regulated by induction of the IL-12 receptor at each division as cells proliferated in response to TCR activation. In contrast, IFN-␣/␤ tended to slow the progression of cell divi...

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IFN-α Is Not Sufficient to Drive Th1 Development Due to Lack of Stable T-bet Expression

Cited by 38 publications

References 62 publications

Reciprocal responsiveness to interleukin-12 and interferon-α specifies human CD8+ effector versus central memory T-cell fates

Reciprocal responsiveness to interleukin-12 and interferon-α specifies human CD8+ effector versus central memory T-cell fates

Multispectral imaging of hematopoietic cells: Where flow meets morphology

IL-12 selectively programs effector pathways that are stably expressed in human CD8+ effector memory T cells in vivo

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