bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of IL-4 Facilitates the Development of Chronic Autoimmune Myasthenia Gravis in C57BL/6 Mice
Authors: Ostlie Norma , Milani Monica , Wang Wei , Okita David , Conti-Fine Bianca M.
Published: April 2014

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4+ T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deficient in IL-4 (IL-4−/−) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4+ T cells respond not only to the TAChR and TAChR α subunit peptides, but also to several mouse AChR α subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR.
IL-4-Producing CD8+ T Cells with a CD62L++(bright) Phenotype Accumulate in a Subgroup of Older Adults and Are Associated with the Maintenance of Intact Humoral Immunity in Old Age
Authors: Schwaiger Susanne , Wolf Anna Maria , Robatscher Peter , Jenewein Brigitte , Grubeck-Loebenstein Beatrix
Published: April 2014

An increased production of proinflammatory cytokines occurs in a high percentage of elderly persons and is associated with an impaired humoral immune response. However, high IL-4 production has also been observed in old age. We now demonstrate an IL-4-producing subpopulation of CD8+ T cells in a subgroup of healthy older adults. This T cell subset is substantial in size and has a characteristic phenotype expressing CD45RO, CD28, CD62L, and CD25. IL-4-producing CD8+ T cells produce large amounts of IL-2 but not IFN-γ or perforin, and these cells do not have a regulatory suppressive effect on other T cells. In vivo IL-4-producing CD8+ T cells can be stably detected over a year. When put into culture they also have a stable cytokine production pattern but fail to produce perforin even in the presence of IL-12. This special T cell type does not occur in persons under the age of 40, but is present in 36% of the persons >60 years of age. In this age group, IL-4-producing CD8+ T cells are more frequent in persons who are still capable of raising a humoral immune response following immunization than in others who fail to produce protective Abs after vaccination. Our results suggest that CD8+ T cells with a CD62L++(bright) phenotype accumulate in a subgroup of older adults. Due to their phenotype that enables them to migrate into lymphoid tissues and to their capacity to produce IL-4, these cells may counterbalance the overproduction of proinflammatory cytokines in old age.
Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study
Authors: Kindzelskii Andrei L. , Petty Howard R.
Published: April 2014

Using high sensitivity fluorescence imaging with shutter speeds ∼600,000 times faster than those of video frames, we have characterized Ca2+ waves within cells in exquisite detail to reveal Ca2+ signaling routes. Polarized neutrophils exhibited a counterclockwise rotating ryanodine-sensitive juxtamembrane Ca2+ wave during temporal calcium spikes. During stimulation with fMLP, a chemotactic factor, two Ca2+ waves traveling in opposite directions around the perimeter of the cell emanated from sites of stimulation (the clockwise wave is verapamil sensitive). Phagocytosed targets exhibit counterclockwise Ca2+ waves traveling about their periphery originating from the plasma membrane. This study: 1) outlines the technology to observe Ca2+ signaling circuitry within small living cells; 2) shows that extracellular spatial information in the form of a chemotactic factor gradient is transduced into intracellular chemical patterns, which provides fresh insights in signaling; 3) suggests that a line of communication exits between the cell surface and phagosomes; and 4) suggests that spatiotemporal Ca2+ patterns contribute to drug actions.
Established T Cell-Driven Germinal Center B Cell Proliferation Is Independent of CD28 Signaling but Is Tightly Regulated Through CTLA-4
Authors: Walker Lucy S. K. , Wiggett Helen E. , Gaspal Fabrina M. C. , Raykundalia Chandra R. , Goodall Margaret D. , Toellner Kai-Michael , Lane Peter J. L.
Published: April 2014

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.
Cutting Edge: The NK Cell Receptor 2B4 Augments Antigen-Specific T Cell Cytotoxicity Through CD48 Ligation on Neighboring T Cells
Authors: Lee Kyung-Mi , Bhawan Sadhna , Majima Takashi , Wei Hairong , Nishimura Michael I. , Yagita Hideo , Kumar Vinay
Published: April 2014

2B4 is expressed on all NK and a subset of memory/effector CD8+ T cells. 2B4 binds to CD48 and activates NK cytotoxicity, but its function on CD8+ T cells is not clear. Furthermore, two isoforms of 2B4 (2B4S and 2B4L) exist in mice but the role of individual isoforms is not known. To address these questions, we generated primary T cell cultures from Ld-specific 2C/Rag2−/− TCR transgenic mice and transduced them with 2B4S or 2B4L. 2B4S- or 2B4L-transduced T cells showed greater cytotoxicity over control cells against CD48+ and CD48− targets, suggesting that ligation of 2B4 by CD48 on target cells was not necessary for 2B4 function. Rather, 2B4/CD48 interaction on adjacent T cells appeared to be critical for cytotoxicity. Therefore, 2B4 functions as a costimulator of CD8+ T cells in MHC-restricted cytotoxicity. We conclude that 2B4/CD48 interactions among T cells themselves can augment CTL lysis of their specific targets.
Cutting Edge: Role of IL-27/WSX-1 Signaling for Induction of T-Bet Through Activation of STAT1 During Initial Th1 Commitment
Authors: Takeda Atsunobu , Hamano Shinjiro , Yamanaka Atsushi , Hanada Toshikatsu , Ishibashi Tatsuro , Mak Tak W. , Yoshimura Akihiko , Yoshida Hiroki
Published: April 2014

WSX-1 is a member of the class I cytokine receptor family with homology to IL-12Rβ2 and is essential for the initial mounting of Th1 responses. STAT1 interacts with tyrosine-phosphorylated WSX-1, and the conserved tyrosine residue of the cytoplasmic domain of WSX-1 is essential for transcriptional activation of STAT1. IL-27 stimulation induced STAT1 phosphorylation in wild-type but not in WSX-1-deficient naive CD4+ T cells. Although IL-27 did not directly induce IFN-γ production by wild-type CD4+ T cells, IL-12-dependent IFN-γ production was augmented by IL-27 stimulation in wild-type naive CD4+ T cells but was impaired in WSX-1-deficient naive CD4+ T cells. Additionally, IL-27 stimulation induced T-bet and IL-12Rβ2 expression in wild-type, but not in WSX-1-deficient, CD4+ T cells. Thus, during the initiation of Th1 differentiation, the IL-27/WSX-1 signaling system plays a pivotal role by STAT1-mediated T-bet induction before the IL-12R system.
Cutting Edge: Dependence of TCR Antagonism on Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Activity
Authors: Kilgore Neely E. , Carter Jenny D. , Lorenz Ulrike , Evavold Brian D.
Published: April 2014

The mechanism by which antagonist peptides inhibit T cell responses is unknown. Mice deficient in Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1) have revealed its importance in the negative regulation of lymphocyte signaling. We investigated a possible role for SHP-1 in T cell antagonism and demonstrate, for the first time, a substantial increase in SHP-1 activity during antagonism of CD4+ T cells. Furthermore, the removal of functional SHP-1 prevents antagonism in these cells. Our data demonstrate that T cell antagonism occurs via a negative intracellular signal that is mediated by SHP-1.
Environmental Chemical-Induced Pro/Pre-B Cell Apoptosis: Analysis of c-Myc, p27Kip1, and p21WAF1 Reveals a Death Pathway Distinct from Clonal Deletion
Authors: Ryu Heui-Young , Mann Koren K. , Schlezinger Jennifer J. , Jensen Brenda , Sherr David H.
Published: April 2014

Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants that suppress the immune system in part by inducing pro/pre-B cell apoptosis. The PAH-induced death signaling pathway resembles the signaling cascade activated during clonal deletion and modeled by B cell receptor cross-linking or by dexamethasone exposure of immature surface Ig+ B cells in that apoptosis is mediated by NF-κB down-regulation. Because a PAH-induced, clonally nonrestricted deletion of B cells would have important implications for B cell repertoire development, the nature of the PAH-induced intracellular death signal was studied further. Particular emphasis was placed on the roles of growth arrest and c-Myc, p27Kip1, and p21WAF1 expression, because all of these elements contribute to clonal deletion. As in clonal deletion models, and as predicted by the down-regulation of NF-κB, PAH-induced death of pro/pre-B cells was at least partially dependent on c-Myc down-regulation. Furthermore, whereas dexamethasone induced a G0/G1 cell cycle arrest, PAH had no effect on pro/pre-B cell growth, indicating that growth arrest and apoptosis occur by separable signaling pathways in this early phase of B cell development. Finally, in contrast to clonal deletion, PAH-induced pro/pre-B cell death was not dependent on p27Kip1 or p21WAF1 up-regulation but did coincide with p53 induction. These results distinguish the PAH-induced apoptosis pathway from that activated during clonal deletion and indicate that signaling cascades leading to growth arrest and/or apoptosis in pro/pre-B cells differ from those active at later B cell developmental stages.
Induction of Tumor Cell Apoptosis In Vivo Increases Tumor Antigen Cross-Presentation, Cross-Priming Rather than Cross-Tolerizing Host Tumor-Specific CD8 T Cells
Authors: Nowak Anna K. , Lake Richard A. , Marzo Amanda L. , Scott Bernadette , Heath William R. , Collins Edward J. , Frelinger Jeffrey A. , Robinson Bruce W. S.
Published: April 2014

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag “sequestration,” or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by ∼80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.
Two Regions of Down-Regulation in the IgE-Mediated Signaling Pathway in Human Basophils
Authors: MacGlashan Donald
Published: April 2014

Previous studies demonstrated that after stimulation of human basophils with a polyclonal anti-IgE Ab, early signaling elements showed sustained phosphorylation, whereas later elements were transient, suggesting that a region of down-regulation involved inhibition of phosphatidylinositol (PI) 3 kinase or its products. However, the current studies show that under some conditions, syk phosphorylation is transient. Generally, stimulation with a variety of Ags makes this early form of down-regulation more apparent. An exploration of the conditions needed to induce early down-regulation indicates that both the nature of aggregation and the cell surface density of IgE play roles. It was also found that the previously described late form of down-regulation (PI3 kinase product transience) can occur in cells displaying early down-regulation (transient syk phosphorylation), but this phenomenon is revealed by testing for subsequent down-regulation of the response to non-cross-reacting stimuli, altering their ability to induce phosphorylation of Akt or extracellular signal-regulated kinase. In contrast, phosphorylation of syk kinase, in response to a non-cross-reacting stimulus, was relatively unaffected by prior stimulation. The magnitude of cross-desensitization of the Akt or extracellular signal-regulated kinase response was a function of the strength of the first stimulus. Mediator release showed a similar cross-desensitization effect. Therefore, stimulation induces two forms of down-regulation, one operating before or at the level of syk phosphorylation, possibly characterizing the process formerly known as specific desensitization, and one that operates in the region of PI3 kinase, accounting for the process formerly known as nonspecific desensitization, which is dependent on the strength of stimulus.
The Lymphoid Past of Mouse Plasmacytoid Cells and Thymic Dendritic Cells
Authors: Corcoran Lynn , Ferrero Isabel , Vremec David , Lucas Karen , Waithman Jason , O’Keeffe Meredith , Wu Li , Wilson Anne , Shortman Ken
Published: April 2014

There has been controversy over the possible lymphoid origin of certain dendritic cell (DC) subtypes. To resolve this issue, DC and plasmacytoid pre-DC isolated from normal mouse tissues were analyzed for transient (mRNA) and permanent (DNA rearrangement) markers of early stages of lymphoid development. About 27% of the DNA of CD8+ DC from thymus, and 22–35% of the DNA of plasmacytoid pre-DC from spleen and thymus, was found to contain IgH gene D-J rearrangements, compared with 40% for T cells. However, the DC DNA did not contain IgH gene V-D-J rearrangements nor T cell Ag receptor β gene D-J rearrangements. The same DC lineage populations containing IgH D-J rearrangements expressed mRNA for CD3 chains, and for pre-Tα. In contrast, little of the DNA of the conventional DC derived from spleen, lymph nodes, or skin, whether CD8+ or CD8−, contained IgH D-J rearrangements and splenic conventional DC expressed very little CD3ε or pre-Tα mRNA. Therefore, many plasmacytoid pre-DC and thymic CD8+ DC have shared early steps of development with the lymphoid lineages, and differ in origin from conventional peripheral DC.
Regulation of CD8+ T Cells Undergoing Primary and Secondary Responses to Infection in the Same Host
Authors: Badovinac Vladimir P. , Messingham Kelly A. Nordyke , Hamilton Sara E. , Harty John T.
Published: April 2014

Naive Ag-specific CD8+ T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8+ T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8+ T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8+ T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8+ T cells present at the time of infection inversely correlated with the magnitude of primary CD8+ T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8+ T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8+ T cell responses was not influenced by the presence of memory CD8+ T cells. However, contraction of secondary CD8+ T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8+ T cell responses against multiple epitopes. The delayed contraction of secondary CD8+ T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8+ T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8+ T cell response.
Prostaglandin D2 Affects the Maturation of Human Monocyte-Derived Dendritic Cells: Consequence on the Polarization of Naive Th Cells
Authors: Gosset Philippe , Bureau Fabrice , Angeli Veronique , Pichavant Muriel , Faveeuw Christelle , Tonnel André-Bernard , Trottein François
Published: April 2014

Among the factors produced at inflammatory sites and those capable of modulating dendritic cell (DC) functions, PGD2 may be important in the outcome of immune responses. The biological roles for PGD2 are in part effected through two plasma membrane G protein-coupled receptors: the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes (CRTH2). In this report, we studied the effects of PGD2 and of its major physiological metabolite, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), on the functions of human monocyte-derived DC. First, we show that PGD2 exerts in vitro chemotactic effects on monocytes via CRTH2 activation while it inhibits the chemokine-driven migration of monocyte-derived DC through DP. We also report that PGD2 and 15d-PGJ2 alter the LPS- and allergen-induced DC maturation and enhance the CD80/CD86 ratio on mature DC in a DP- and CRTH2-independent manner. Moreover, PGD2 and 15d-PGJ2 strongly reduce the secretion of the Th1 promoting cytokine IL-12 and affect the synthesis of chemokines involved in Th1 cell chemotaxis, particularly CXCL10. Inhibition of cytokine/chemokine secretion implicates at least in part DP, but not CRTH2. The effects exerted by PGD2 are associated with the phosphorylation of CREB, but do not parallel with the deactivation of the NF-κB and mitogen-activated protein kinase pathways. In contrast, 15d-PGJ2 seems to target other cellular proteins. Finally, in a model of Th CD45RA+ differentiation induced by allergen- and superantigen-pulsed DC, PGD2 impacts on the orientation of the immune response by favoring a Th2 response.
Differential Roles of CB1 and CB2 Cannabinoid Receptors in Mast Cells
Authors: Samson Maria-Teresa , Small-Howard Andrea , Shimoda Lori M. N. , Koblan-Huberson Murielle , Stokes Alexander J. , Turner Helen
Published: April 2014

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via FcεRI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.
Stochastic Model of T Cell Proliferation: A Calculus Revealing IL-2 Regulation of Precursor Frequencies, Cell Cycle Time, and Survival
Authors: Deenick Elissa K. , Gett Amanda V. , Hodgkin Philip D.
Published: April 2014

The outcome of Ag exposure is dictated by complex regulation of T cell proliferation. The rates of proliferation and survival are altered by numerous signals that the cell receives and integrates to achieve a net response. We have illustrated previously how small changes in kinetic parameters can lead to large differences, even under conditions of saturating IL-2. In this study, we examine the effect of varying IL-2 concentration on T cell response and develop a model incorporating additional parameters of proliferation and survival. Strikingly, the proportion of cells that enter the first division, but not the time at which they enter, is dramatically altered by IL-2. Furthermore, the survival and average division time of cells in later divisions are also altered by IL-2 concentration. Together, the small simultaneous effects on these parameters result in large differences in total cell number. These results reveal how in vitro systems may exaggerate the contribution of IL-2, and thus how costimuli or additional helper cells that alter IL-2 concentration, even by relatively small amounts, will generate large in vitro differences in cell number and therefore appear obligatory. Furthermore, they illustrate how a quantitative model of T cell activation can clarify how complex signal integration is handled by T cells in situ, and therefore more appropriately aid development of a theory of behavior.
Active Form of Notch Members Can Enforce T Lymphopoiesis on Lymphoid Progenitors in the Monolayer Culture Specific for B Cell Development
Authors: Hozumi Katsuto , Abe Natsumi , Chiba Shigeru , Hirai Hisamaru , Habu Sonoko
Published: April 2014

The in vitro induction of T lymphopoiesis needs the precise stereoscopic structure of thymus tissues as seen in fetal thymus organ culture. In this study, we demonstrated for the first time that the introduction of the intracellular region of Notch1 can induce T cells expressing TCR without any thymic environment. In the coculture on the monolayer of OP-9, which was originally known to support B cell specific development, hemopoietic progenitors developed into Thy-1+CD25+ T lineage cells if the progenitor cells were infected with the retrovirus containing Notch1 intracellular domains. The Thy-1+ cells progressed to a further developmental stage, CD4 and CD8 double-positive cells expressing TCR on the cell surface, if they were further cultured on OP-9 or in the thymus. However, T cell induction by intracellular Notch1 failed unless both OP-9 and IL-7 were present. It is notable that Notch2 and Notch3 showed an effect on T lymphopoiesis similar to that of Notch1. These results indicate that in vitro T lymphopoiesis is inducible by signaling via Notch family members in a lineage-specific manner but shares other stroma-derived factors including IL-7 with B lymphopoiesis.
Histone Deacetylation, But Not Hypermethylation, Modifies Class II Transactivator and MHC Class II Gene Expression in Squamous Cell Carcinomas
Authors: Kanaseki Takayuki , Ikeda Hideyuki , Takamura Yukio , Toyota Minoru , Hirohashi Yoshihiko , Tokino Takashi , Himi Tetsuo , Sato Noriyuki
Published: April 2014

In this study, we first categorized nine squamous cell carcinoma (SCC) cell lines into two groups in terms of the expression of HLA-DR, -DP, and -DQ molecules. Subsequently, the expression of class II transactivator (CIITA) was studied in these cell lines, because it is widely accepted that the expression of MHC class II molecules is regulated by different types of CIITA transcripts that are initiated by distinct promoters. The majority of the SCC cell lines (six of nine) expressed HLA-DR molecules and CIITA promoter IV (pIV) transcripts in the presence of IFN-γ. In contrast, three of the nine SCC cell lines were completely negative for class II molecules and all types of CIITA, suggesting epigenetic changes in the promoter region in these cells. Previously, methylation of CIITA pIV was reported to silence CIITA gene expression. We extensively studied the methylation status of CIITA pIV using a panel of 22 SCC cell lines. Remarkably, none of the SCC cell lines demonstrated hypermethylation at the site. In contrast, treatment with a histone deacetylation inhibitor in combination with IFN-γ clearly restored the expression of the CIITA type IV gene in the HLA-DR-negative SCC cell lines, and the acetylation status of histone H3 examined by chromatin immunoprecipitation analysis was closely associated with the gene expression. Moreover, stable transfection of the CIITA gene into an HLA-DR-negative cell line restored constitutive expression of MHC class II molecules. Therefore, histone deacetylation, but not hypermethylation, modifies CIITA DNA and class II gene expression in SCC.
Differential Control of Cell Cycle, Proliferation, and Survival of Primary T Lymphocytes by Purine and Pyrimidine Nucleotides
Authors: Quéméneur Laurence , Gerland Luc-Marie , Flacher Monique , Ffrench Martine , Revillard Jean-Pierre , Genestier Laurent
Published: April 2014

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G1 to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.
Mechanisms of Accelerated Immune-Mediated Diabetes Resulting from Islet β Cell Expression of a Fas Ligand Transgene
Authors: Silva Diego G. , Petrovsky Nikolai , Socha Luis , Slattery Robyn , Gatenby Paul , Charlton Brett
Published: April 2014

Nonobese diabetic (NOD) mice transgenic for Fas ligand (FasL) on islet β cells (HIPFasL mice) exhibit an accelerated diabetes distinct from the normal autoimmune diabetes of NOD mice. This study was undertaken to define the mechanism underlying accelerated diabetes development in HIPFasL mice. It was found that diabetes in HIPFasL mice is dependent on the NOD genetic background, as HIPFasL does not cause diabetes when crossed into other mice strains and is lymphocyte dependent, as it does not develop in HIPFasLSCID mice. Diabetes development in NODSCID recipients of diabetic HIPFasL splenocytes is slower than when using splenocytes from diabetic NOD mice. β cells from HIPFasL mice are more susceptible to cytokine-induced apoptosis than wild-type NOD β cells, and this can be blocked with anti-FasL Ab. HIPFasL islets are more rapidly destroyed than wild-type islets when transplanted into nondiabetic NOD mice. This confirms that FasL+ islets do not obtain immune privilege, and instead NOD β cells constitutively expressing FasL are more susceptible to apoptosis induced by Fas-FasL interaction. These findings are consistent with the accelerated diabetes of young HIPFasL mice being a different disease process from the autoimmune diabetes of wild-type NOD mice. The data support a mechanism by which cytokines produced by the insulitis lesion mediate up-regulation of β cell Fas expression, resulting in suicide or fratricide of HIPFasL β cells that overexpress FasL.
IL-18 and IL-12 Signal Through the NF-κB Pathway to Induce NK-1R Expression on T Cells
Authors: Weinstock Joel V. , Blum Arthur , Metwali Ahmed , Elliott David , Arsenescu Razvan
Published: April 2014

Substance P engages the T cell neurokinin 1 receptor (NK-1R) to enhance IFN-γ production. NK-1R on T cells is inducible. We studied mechanisms regulating T cell NK-1R expression. Murine splenocytes were cultured for 4 h with or without rIL-12 or rIL-18. Both IL-12 and IL-18 induced splenic T cells to express NK-1R transcripts. Induction was blocked by actinomycin D, but not cycloheximide, suggesting that protein synthesis was not required for initiation of NK-1R gene transcription. Inhibition of T cell NF-κB activation or NF-κB nuclear translocation also blocked NK-1R transcription. IL-12 and IL-18 strongly induce NK-1R mRNA expression in splenocytes from Stat4−/− mice, suggesting that the Stat4 pathway was not required for the induction of NK-1R transcription. Splenic T cells exposed to IL-12 or IL-18 in the presence of IL-10 expressed no NK-1R mRNA. However, TGFβ did not prevent NK-1R mRNA expression. Thus, IL-12 and IL-18 induce T cells to express NK-1R through NF-κB activation. IL-10, a regulator of the Th1 response, blocks this activation. These data further suggest that SP and NK-1R, which promote IFN-γ synthesis, are part of the Th1 pathway of immunity.
Superantigen-Induced Regulatory T Cells Display Different Suppressive Functions in the Presence or Absence of Natural CD4+CD25+ Regulatory T Cells In Vivo
Authors: Grundström Susanna , Cederbom Lukas , Sundstedt Anette , Scheipers Peter , Ivars Fredrik
Published: April 2014

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4+CD25+ regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4+CD25+ regulatory T cells. In this study, we demonstrate that CD4+CD25+ as well as CD4+CD25− T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4+CD25+ regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4+ T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4+CD25− T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.
IL-15 Promotes the Survival of Naive and Memory Phenotype CD8+ T Cells
Authors: Berard Marion , Brandt Katja , Paus Silvia Bulfone , Tough David F.
Published: April 2014

IL-15 stimulates the proliferation of memory phenotype CD44highCD8+ T cells and is thought to play a key role in regulating the turnover of these cells in vivo. We have investigated whether IL-15 also has the capacity to affect the life span of naive phenotype (CD44low) CD8+ T cells. We report that IL-15 promotes the survival of both CD44low and CD44high CD8+ T cells, doing so at much lower concentrations than required to induce proliferation of CD44high cells. Rescue from apoptosis was associated with the up-regulation of Bcl-2 in both cell types, whereas elevated expression of Bcl-xL was observed among CD44high but not CD44low CD8+ cells. An investigation into the role of IL-15R subunits in mediating the effects of IL-15 revealed distinct contributions of the α- and β- and γ-chains. Most strikingly, IL-15Rα was not essential for either induction of proliferation or promotion of survival by IL-15, but did greatly enhance the sensitivity of cells to low concentrations of IL-15. By contrast, the β- and γ-chains of the IL-15R were absolutely required for the proliferative and pro-survival effects of IL-15, although it was not necessary for CD44highCD8+ cells to express higher levels of IL-15Rβ than CD44low cells to proliferate in response to IL-15. These results show that IL-15 has multiple effects on CD8 T cells and possesses the potential to regulate the life span of naive as well as memory CD8+ T cells.
A Peptide from Heat Shock Protein 60 Is the Dominant Peptide Bound To Qa-1 in the Absence of the MHC Class Ia Leader Sequence Peptide Qdm
Authors: Davies Adrian , Kalb Suzanne , Liang Bitao , Aldrich Carla J. , Lemonnier François A. , Jiang Hong , Cotter Robert , Soloski Mark J.
Published: April 2014

The MHC class Ib molecule Qa-1 binds specifically and predominantly to a single 9-aa peptide (AMAPRTLLL) derived from the leader sequence of many MHC class Ia proteins. This peptide is referred to as Qdm. In this study, we report the isolation and sequencing of a heat shock protein 60-derived peptide (GMKFDRGYI) from Qa-1. This peptide is the dominant peptide bound to Qa-1 in the absence of Qdm. A Qa-1-restricted CTL clone recognizes this heat shock protein 60 peptide, further verifying that it binds to Qa-1 and a peptide from the homologous Salmonella typhimurium protein GroEL (GMQFDRGYL). These observations have implications for how Qa-1 can influence NK cell and T cell effector function via the TCR and CD94/NKG2 family members, and how this effect can change under conditions that cause the peptides bound to Qa-1 to change.
Extracellular Matrix Conditions T Cells for Adhesion to Tissue Interstitium
Authors: Krivacic Kimberly A. , Levine Alan D.
Published: April 2014

The activation and differentiation of peripheral blood T cells (PBT) are known to correlate with increased surface expression and adhesive capacity of β1 integrins, which mediate adhesion to the extracellular matrix (ECM). However, little is known about the regulation of integrin expression, affinity, and avidity on tissue T cells after they are embedded in the interstitial ECM. In this study we show that tissue T cells, freshly isolated from their residence in the interstitial ECM of the intestinal lamina propria, express a distinct subset of functionally active integrins that contribute to enhanced adhesion to purified collagen, fibronectin, and cell-derived ECM when compared with freshly isolated, short term activated, and long term cultured PBT. Furthermore, integrin usage is distinct between circulating and tissue-derived T cells, in that lamina propria T cells prefer to bind to collagen, while PBT lymphoblasts choose fibronectin when presented with a complex, three-dimensional, cell-derived matrix. To identify the extrinsic factors that regulate the conversion from a nonadhesive PBT to highly adhesive tissue T cell, we demonstrate that activation of PBT in the presence of fibronectin or collagen rapidly generates a surface integrin expression profile, an integrin usage pattern, and adhesive capacity mirroring that of a tissue T cell. These results indicate that the tissue ECM microenvironment instructs newly arrived T cells for further interactions with the underlying matrix and thereby imprints them with a signature tissue adhesive phenotype.
Mast Cells Express Novel Functional IL-15 Receptor α Isoforms
Authors: Bulanova Elena , Budagian Vadim , Orinska Zane , Krause Hans , Paus Ralf , Bulfone-Paus Silvia
Published: April 2014

Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15Rα chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15Rα gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15RαΔ4, IL-15RαΔ3,4, IL-15RαΔ3,4,5). After transient transfection in COS-7 cells, all IL-15Rα isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15Rα isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15Rα isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15Rα isoforms, which can explain the selective regulatory effects of IL-15 on these cells.
Attenuation of Immunological Symptoms of Allergic Asthma in Mice Lacking the Tyrosine Kinase ITK
Authors: Mueller Cynthia , August Avery
Published: April 2014

Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, TH2 cells, and cytokines, increased mucous production in the lung, and elevated serum IgE. This TH2-type response suggests a prominent role for TH2 cells and their cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown to play a role in the differentiation and/or function of TH2-type cells, suggesting that ITK may represent a good target for the control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immunological symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice lacking ITK. T cells from ITK−/− mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13, in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the TH2 cytokines IL-5 and IL-13 and may be an attractive target for antiasthmatic drugs.
Nitric Oxide-Independent CTL Suppression during Tumor Progression: Association with Arginase-Producing (M2) Myeloid Cells
Authors: Liu Yuanqing , Van Ginderachter Jo A. , Brys Lea , De Baetselier Patrick , Raes Geert , Geldhof Anja B.
Published: April 2014

Most of the mice bearing a s.c. BW-Sp3 lymphoma tumor mount a CD8+ T cell-mediated response resulting in tumor regression. Nonetheless, tumor progression occurs in some of the recipients and is associated with CTL inactivity. We demonstrated that T cell-activating APC were induced in regressors whereas T cell suppressive myeloid cells predominated in the spleen of progressors. Indeed, in vitro depletion of either the adherent or the CD11b+ populations restored T cell cytotoxicity and proliferation in these mice. This CTL inhibition was cell-to-cell contact-dependent but not mediated by NO. However, the same progressor suppressive cells prevented the activity of in vitro-restimulated CTLs derived from regressors in a cell-to-cell contact and NO-dependent fashion. Thus, either the NO-dependent or -independent suppressive pathway prevailed, depending on the target CTL population. In addition, the suppressive population expressed a high arginase activity, suggesting an association of the suppressive phenotype with alternatively activated (M2) myeloid cells. However, the high arginase activity is not directly involved in the suppressive process. Our results provide new insights for myeloid cell-mediated CTL inhibition during cancer progression.
Early IL-2 Production by Mouse Dendritic Cells Is the Result of Microbial-Induced Priming
Authors: Granucci Francesca , Feau Sonia , Angeli Véronique , Trottein François , Ricciardi-Castagnoli Paola
Published: April 2014

Dendritic cells (DCs) are professional APCs able to initiate innate and adaptive immune responses against invading pathogens. Different properties such as the efficient Ag processing machinery, the high levels of expression of costimulatory molecules and peptide-MHC complexes, and the production of cytokines contribute in making DCs potent stimulators of naive T cell responses. Recently we have observed that DCs are able to produce IL-2 following bacterial stimulation, and we have demonstrated that this particular cytokine is a key molecule conferring to early bacterial activated DCs unique T cell priming capacity. In the present study we show that many different microbial stimuli, but not inflammatory cytokines, are able to stimulate DCs to produce IL-2, indicating that DCs can distinguish a cytokine-mediated inflammatory process from the actual presence of an infection. The capacity to produce IL-2 following a microbial stimuli encounter is a feature shared by diverse DC subtypes in vivo, such as CD8α+ and CD8α− splenic DCs and epidermal Langerhans cells. When early activated DCs interact with T cells, IL-2 produced by DCs is enriched at the site of cell-cell contact, confirming the importance of DCs-derived IL-2 in T cell activation.
TNF Enhances CD4+ T Cell Alloproliferation, IFN-γ Responses, and Intestinal Graft-Versus-Host Disease by IL-12-Independent Mechanisms
Authors: Brown Geri R. , Lee Edward L. , Thiele Dwain L.
Published: April 2014

Inhibition of TNF/TNFR2 interactions ameliorates intestinal graft-vs-host disease (GVHD) and Th1 cytokine responses induced by transfer of B6 CD4+ spleen cells into irradiated MHC class II disparate B6.C-H-2bm12 (bm12) × B6 F1 recipients. The present studies examined whether these effects of TNF are IL-12 dependent. T cell proliferative responses of B6.129S1-IL-12rb2tm1Jm (B6.IL-12R−/−) responder spleen cells were found to be comparable to those of control B6 spleen cells. TNF inhibition reduced T cell proliferation and IFN-γ production in supernatants of MLC using either B6.IL-12R−/− or control B6 responder cells. GVHD induced wasting disease in recipients of B6.IL-12R−/− CD4+ spleen cells that received a TNF inhibitor-encoding adenovirus (5.4 ± 6.5% weight loss (n = 7)) was significantly reduced compared with levels of weight loss observed in recipients that had received a control adenovirus (25.7 ± 12.2% weight loss (n = 11), p = 0.001). Furthermore, TNF inhibition was associated with a reduction in colonic GVHD scores (p = 0.039) and in the percentage of the splenic CD4+ T cells that expressed IFN-γ (16 vs 6%). These findings indicate that TNF promotes CD4+ T cell alloproliferation, IFN-γ responses, and intestinal GVHD by IL-12-independent mechanisms.
A Subset of Human Dendritic Cells in the T Cell Area of Mucosa-Associated Lymphoid Tissue with a High Potential to Produce TNF-α
Authors: de Baey Annegret , Mende Ines , Baretton Gustavo , Greiner Axel , Hartl Wolfgang H. , Baeuerle Patrick A. , Diepolder Helmut M.
Published: April 2014

Recently, a new class of human dendritic cell (DC) precursors has been described in the peripheral blood recognized by the mAb M-DC8. These cells represent ∼1% of PBMC and acquire several characteristics of myeloid DC upon in vitro culture. In this report we show that M-DC8+ monocytes secrete in response to LPS >10 times the amount of TNF-α as M-DC8− monocytes, but produce significantly less IL-10. Consistent with a role in inflammatory responses, we found that M-DC8+ cells localized in the T cell area of inflamed human tonsils and in the subepithelial dome region of Peyer’s patches. In patients with active Crohn’s disease, abundant M-DC8+ cells were detectable in inflamed ileal mucosa, which were entirely depleted after systemic steroid treatment. Our results indicate that M-DC8+ cells are cells of DC phenotype in inflamed mucosa-associated lymphoid tissue that may contribute to the high level of TNF-α production in Crohn’s disease. We infer that selective elimination of M-DC8+ cells in inflammatory diseases has therapeutic potential.
Repertoire Shift in the Humoral Response to Phosphocholine-Keyhole Limpet Hemocyanin: VH Somatic Mutation in Germinal Center B Cells Impairs T15 Ig Function
Authors: Wiens Gregory D. , Brown McKay , Rittenberg Marvin B.
Published: April 2014

Phosphocholine (PC) is a naturally occurring Ag common to many pathogenic microorganisms. Early in the primary response to PC conjugated to keyhole limpet hemocyanin (KLH), T15 Id+ Abs constitute >90% of the serum Ig in BALB/c mice. During the late primary and memory response to PC-protein, a shift in the repertoire occurs and T15 Id+ Abs lose dominance. In this study, we use immunohistochemistry and single germinal center microdissection to locate T15 Id+ cells in the spleen in a primary response to PC-KLH. We demonstrate T15 Id+ B cells and VH1-DFL16.1-JH1 and Vκ22-Jκ5 rearrangements in germinal centers early in the immune response; thus loss of T15 dominance is not due to lack of T15 cells within germinal centers. One-hundred thirty one VH1 and 57 Vκ22 rearrangements were cloned and sequenced. Thirty four percent of the VH1 clones and 37% of the Vκ22 clones contained somatic mutations indicating participation in the germinal center response. Six variant T15 H clones were expressed with wild-type T15 L chain in vitro. Two of these Abs were defective in secretion providing the first evidence that mutation occurring in vivo can disrupt Ig assembly and secretion. Of the four secretion-competent Abs, two failed to display binding to PC-protein, while the other two displayed altered carrier recognition. These results indicate that somatic mutation of T15 in vivo can result in the loss of binding and secretion, potentially leading to B cell wastage. The failure of T15 to gain affinity enhancing mutations in the face of these detrimental changes may contribute to repertoire shift.