bluuebunny/crossref_metadata_2025 huggingface dataset:
Soluble HLA-G Inhibits Cell Cycle Progression in Human Alloreactive T Lymphocytes
Authors: Bahri Rajia , Hirsch Francois , Josse Adeline , Rouas-Freiss Nathalie , Bidere Nicolas , Vasquez Aime , Carosella Edgardo D. , Charpentier Bernard , Durrbach Antoine
Published: April 2014

HLA-G is involved in regulating T cell responses. Various mechanisms have been proposed to explain the inhibition of T cell proliferation. In this context, the possible role of HLA-G in cell cycle regulation remains to be explored. Using stably transfected M8 cells expressing the secreted isoform (HLA-G5) of HLA-G, we investigated the role of HLA-G in inducing apoptosis and in controlling the cell cycle of activated T cells. Soluble HLA-G (HLA-G5) inhibited both CD4 and CD8 T cell proliferation. However, HLA-G5 did not induce T cell apoptosis, as determined by 3,3′-diethyloxacarbocyanine and propidium iodine labeling. It induced accumulation of the retinoblastoma protein, but not its phosphorylated and active form. Treatment of activated T cells with HLA-G5 also reduced the amounts of cyclin D2, E, A, and B by >80%. In contrast, it induced an accumulation of p27kip, but not p21cip, in activated T cells. HLA-G does not induce apoptosis of alloreactive T cells, but induces p27kip1 and inhibits cell cycle progression.
The Endoplasmic Reticulum Is a Key Component of the Plasma Cell Death Pathway
Authors: Pelletier Nadège , Casamayor-Pallejà Montserrat , De Luca Karelle , Mondière Paul , Saltel Frédéric , Jurdic Pierre , Bella Chantal , Genestier Laurent , Defrance Thierry
Published: April 2014

Plasma cells (PC) are the effector cells of the humoral Ab response. Unlike other dedicated secretory cells, they exist as two populations with opposite cell fates: short-lived and long-lived PC. Upon transformation they lead to an incurable neoplasia called multiple myeloma. In this study we have explored the molecular mechanism of PC death. Our data show that their apoptotic pathway is unique among other hemopoietic cells inasmuch as neither the death receptors nor the mitochondria play the central role. PC apoptosis is initiated by activation of Bax at the endoplasmic reticulum membrane and subsequent activation of the endoplasmic reticulum-associated caspase-4 before the release of mitochondrial apoptogenic factors. Together, our observations indicate that the cardinal function of PC (i.e., Ig secretion) is also the cause of their death.
Direct Costimulatory Effect of TLR3 Ligand Poly(I:C) on Human γδ T Lymphocytes
Authors: Wesch Daniela , Beetz Susann , Oberg Hans-Heinrich , Marget Matthias , Krengel Kirsten , Kabelitz Dieter
Published: April 2014

TLR3 recognizes viral dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). TLR3 expression is commonly considered to be restricted to dendritic cells, NK cells, and fibroblasts. In this study we report that human γδ and αβ T lymphocytes also express TLR3, as shown by quantitative real-time PCR, flow cytometry, and confocal microscopy. Although T cells did not respond directly to poly(I:C), we observed a dramatic increase in IFN-γ secretion and an up-regulation of CD69 when freshly isolated γδ T cells were stimulated via TCR in the presence of poly(I:C) without APC. IFN-γ secretion was partially inhibited by anti-TLR3 Abs. In contrast, poly(I:C) did not costimulate IFN-γ secretion by αβ T cells. These results indicate that TLR3 signaling is differentially regulated in TCR-stimulated γδ and αβ T cells, suggesting an early activation of γδ T cells in antiviral immunity.
Flightless I Homolog Negatively Modulates the TLR Pathway
Authors: Wang Tianyi , Chuang Tsung-Hsien , Ronni Tapani , Gu Sheng , Du Yu-Chun , Cai Hong , Sun Hui-Qiao , Yin Helen L. , Chen Xian
Published: April 2014

To date, much of our knowledge about the signaling networks involved in the innate immune response has come from studies using nonphysiologic model systems rather than actual immune cells. In this study, we used a dual-tagging proteomic strategy to identify the components of the MyD88 signalosome in murine macrophages stimulated with lipid A. This systems approach revealed 16 potential MyD88-interacting partners, one of which, flightless I homolog (Fliih) was verified to interact with MyD88 and was further characterized as a negative regulator of the TLR4-MyD88 pathway. Conversely, a reduction in endogenous Fliih by small-interfering RNA enhanced the activation of NF-κB, as well as cytokine production by LPS. Results from immunoprecipitation and a two-hybrid assay further indicated that Fliih directly interfered with the formation of the TLR4-MyD88 signaling complex. These results in turn suggest a new basis for the regulation of the TLR pathway by Fliih.
Immunologic Control of Tumors by In Vivo Fcγ Receptor-Targeted Antigen Loading in Conjunction with Double-Stranded RNA-Mediated Immune Modulation
Authors: Bot Adrian , Smith Dan , Phillips Bill , Bot Simona , Bona Constantin , Zaghouani Habib
Published: April 2014

Despite the expression of non-self or neo-epitopes, many tumors such as lymphoid malignancies or cancers induced by oncogenic viruses are able to gradually overcome the immune defense mechanisms and spread. Using a preclinical model of hematological malignancy, we show that Ig-associated idiotypic determinants are recognized by the immune system in a fashion that results in immune deviation, allowing tumor progression and establishment of metastases. Using gene-targeted mice, we show that anti-idiotypic MHC class I-restricted immunity is promoted by ITAM motif (ITAM+) FcγR, but kept in check by ITIM motif (ITIM+) FcγRIIB-mediated mechanisms. In addition to interfering with the functionality of ITIM+ FcγR, effective anti-idiotypic and antitumoral immunity can be achieved by FcγR-targeted delivery of epitope in conjunction with administration of stimulatory motifs such as dsRNA, correcting the ineffective response to idiotypic epitopes. The immune process initiated by FcγR-mediated targeting of epitope together with dsRNA, resulted in control of tumor growth, establishment of immune memory and protection against tumors bearing antigenic variants. In summary, targeted delivery of MHC class I-restricted epitopes via ITAM+ FcγR, in conjunction with use of TLR-binding immune stimulatory motifs such as dsRNA, overcomes suboptimal responses to idiotypic determinants and may constitute a novel approach for the treatment of a broad range of malignancies. Finally, the results shed light on the mechanisms regulating the idiotypic network and managing the diversity associated with immune receptors.
Murine Mammary Carcinoma Exosomes Promote Tumor Growth by Suppression of NK Cell Function
Authors: Liu Cunren , Yu Shaohua , Zinn Kurt , Wang Jianhua , Zhang Liming , Jia Yujiang , Kappes John C. , Barnes Stephen , Kimberly Robert P. , Grizzle William E. , Zhang Huang-Ge
Published: April 2014

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.
Potentiation of Antigen-Stimulated Vγ9Vδ2 T Cell Cytokine Production by Immature Dendritic Cells (DC) and Reciprocal Effect on DC Maturation
Authors: Devilder Marie-Claire , Maillet Sophie , Bouyge-Moreau Isabelle , Donnadieu Emmanuel , Bonneville Marc , Scotet Emmanuel
Published: April 2014

Vγ9Vδ2 T cells, a major γδ PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vγ9Vδ2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vγ9Vδ2 T cell clones and ex vivo memory Vγ9Vδ2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vγ9Vδ2 production of inflammatory cytokines required for their own maturation suggested that Vγ9Vδ2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vγ9Vδ2 cells induced accelerated maturation of Ag-expressing iDC but not “bystander” DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vγ9Vδ2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vγ9Vδ2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vγ9Vδ2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.
Critical, but Conditional, Role of OX40 in Memory T Cell-Mediated Rejection
Authors: Vu Minh Diem , Clarkson Michael R. , Yagita Hideo , Turka Laurence A. , Sayegh Mohamed H. , Li Xian Chang
Published: April 2014

Memory T cells can be a significant barrier to the induction of transplant tolerance. However, the molecular pathways that can regulate memory T cell-mediated rejection are poorly defined. In the present study we tested the hypothesis that the novel alternative costimulatory molecules (i.e., ICOS, 4-1BB, OX40, or CD30) may play a critical role in memory T cell activation and memory T cell-mediated rejection. We found that memory T cells, generated by either homeostatic proliferation or donor Ag priming, induced prompt skin allograft rejection regardless of CD28/CD154 blockade. Phenotypic analysis showed that, in contrast to naive T cells, such memory T cells expressed high levels of OX40, 4-1BB, and ICOS on the cell surface. In a skin transplant model in which rejection was mediated by memory T cells, blocking the OX40/OX40 ligand pathway alone did not prolong the skin allograft survival, but blocking OX40 costimulation in combination with CD28/CD154 blockade induced long-term skin allograft survival, and 40% of the recipients accepted their skin allograft for >100 days. In contrast, blocking the ICOS/ICOS ligand and the 4-1BB/4-1BBL pathways alone or combined with CD28/CD154 blockade had no effect in preventing skin allograft rejection. OX40 blockade did not affect the homeostatic proliferation of T cells in vivo, but markedly inhibited the effector functions of memory T cells. Our data demonstrate that memory T cells resisting to CD28/CD154 blockade in transplant rejection are sensitive to OX40 blockade and suggest that OX40 is a key therapeutic target in memory T cell-mediated rejection.
CD40 Expression by Microglial Cells Is Required for Their Completion of a Two-Step Activation Process during Central Nervous System Autoimmune Inflammation
Authors: Ponomarev Eugene D. , Shriver Leah P. , Dittel Bonnie N.
Published: April 2014

Microglial cells are monocytic lineage cells that reside in the CNS and have the capacity to become activated during various pathological conditions. Although it was demonstrated that activation of microglial cells could be achieved in vitro by the engagement of CD40-CD40L interactions in combination with proinflammatory cytokines, the exact factors that mediate activation of microglial cells in vivo during CNS autoimmunity are ill-defined. To investigate the role of CD40 in microglial cell activation during experimental autoimmune encephalomyelitis (EAE), we used bone marrow chimera mice that allowed us to distinguish microglial cells from peripheral macrophages and render microglial cells deficient in CD40. We found that the first step of microglial cell activation was CD40-independent and occurred during EAE onset. The first step of activation consisted of microglial cell proliferation and up-regulation of the activation markers MHC class II, CD40, and CD86. At the peak of disease, microglial cells underwent a second step of activation, which was characterized by a further enhancement in activation marker expression along with a reduction in proliferation. The second step of microglial cell activation was CD40-dependent and the failure of CD40-deficient microglial cells to achieve a full level of activation during EAE was correlated with reduced expansion of encephalitogenic T cells and leukocyte infiltration in the CNS, and amelioration of clinical symptoms. Thus, our findings demonstrate that CD40 expression on microglial cells is necessary to complete their activation process during EAE, which is important for disease progression.
Soluble Form of T Cell Ig Mucin 3 Is an Inhibitory Molecule in T Cell-Mediated Immune Response
Authors: Geng Hui , Zhang Gui-Mei , Li Dong , Zhang Hui , Yuan Ye , Zhu Han-Gang , Xiao Han , Han Ling-Fei , Feng Zuo-Hua
Published: April 2014

T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-γ in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.
A Cell-Type Specific CD1d Expression Program Modulates Invariant NKT Cell Development and Function
Authors: Zimmer Michael I. , Colmone Angela , Felio Kyrie , Xu Honglin , Ma Averil , Wang Chyung-Ru
Published: April 2014

Invariant NK T (iNKT) cells are a distinct subset of T cells that rapidly produce an array of immunoregulatory cytokines upon activation. Cytokines produced by iNKT cells subsequently transactivate other leukocytes and elicit their respective effector functions. In this way, iNKT cells play a central role in coordinating the development of immune responses in a variety of settings. However, the mechanisms governing the quality of the iNKT cell response elicited remain poorly defined. To address whether changes in the CD1d expression pattern could regulate iNKT cell function, we generated a transgenic (Tg) mouse model in which thymocytes and peripheral T cells express high levels of CD1d (Lck-CD1d Tg+ mice). The expression of CD1d by T cells was sufficient to rescue development of iNKT cells in mice deficient of endogenous CD1d. However, the relative proportions of iNKT cell subsets in Lck-CD1d Tg+ mice were distinctly different from those in wild-type mice, suggesting an altered developmental program. Additionally, iNKT cells were hyporesponsive to antigenic stimulation in vivo. Interestingly, Lck-CD1d Tg+ mice develop liver pathology in the absence of any exogenous manipulation. The results of these studies suggest that changes to the CD1d expression program modulate iNKT cell development and function.
Protein Expression of TNF-α in Psoriatic Skin Is Regulated at a Posttranscriptional Level by MAPK-Activated Protein Kinase 2
Authors: Johansen Claus , Funding Anne Toftegaard , Otkjaer Kristian , Kragballe Knud , Jensen Uffe Birk , Madsen Mogens , Binderup Lise , Skak-Nielsen Tine , Fjording Marianne Scheel , Iversen Lars
Published: April 2014

Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-α protein expression, but similar TNF-α mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-α expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1β caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-α protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1β. This increase in TNF-α protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-α, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-α protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.
Human Naive CD8 T Cells Down-Regulate Expression of the WNT Pathway Transcription Factors Lymphoid Enhancer Binding Factor 1 and Transcription Factor 7 (T Cell Factor-1) following Antigen Encounter In Vitro and In Vivo
Authors: Willinger Tim , Freeman Tom , Herbert Mark , Hasegawa Hitoshi , McMichael Andrew J. , Callan Margaret F. C.
Published: April 2014

The transcription factors lymphoid enhancer binding factor 1 (LEF1) and transcription factor 7 (TCF7) (T cell factor-1 (TCF-1)) are downstream effectors of the WNT signaling pathway, which is a critical regulator of T cell development in the thymus. In this study, we show that LEF1 and TCF7 (TCF-1) are not only expressed in thymocytes, but also in mature T cells. Our data demonstrate that Ag encounter in vivo and engagement of the TCR or IL-15 receptor in vitro leads to the down-regulation of LEF1 and TCF7 (TCF-1) expression in human naive CD8 T cells. We further show that resting T cells preferentially express inhibitory LEF1 and TCF7 (TCF-1) isoforms and that T cell activation changes the isoform balance in favor of stimulatory TCF7 (TCF-1) isoforms. Altogether, our study suggests that proteins involved in the WNT signaling pathway not only regulate T cell development, but also peripheral T cell differentiation.
Tumor-Specific CD4+ T Cells Are Activated by “Cross-Dressed” Dendritic Cells Presenting Peptide-MHC Class II Complexes Acquired from Cell-Based Cancer Vaccines
Authors: Dolan Brian P. , Gibbs Kenneth D. , Ostrand-Rosenberg Suzanne
Published: April 2014

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through “cross-dressing.” Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.
CXCR3/CXCR3 Ligand Biological Axis Impairs RENCA Tumor Growth by a Mechanism of Immunoangiostasis
Authors: Pan Judong , Burdick Marie D. , Belperio John A. , Xue Ying Ying , Gerard Craig , Sharma Sherven , Dubinett Steven M. , Strieter Robert M.
Published: April 2014

Metastatic renal cell carcinoma (RCC) responds poorly to chemo- or radiation therapy but appears to respond to systemic immunotherapy (i.e., IL-2 and/or IFN-α), albeit with only 5–10% durable response. The CXCR3/CXCR3 ligand biological axis plays an important role in mediating type 1 cytokine-dependent cell-mediated immunity, which could be beneficial for attenuating RCC if optimized. We found that systemic IL-2 induced the expression of CXCR3 on circulating mononuclear cells but impaired the CXCR3 ligand chemotactic gradient from plasma to tumor by increasing circulating CXCR3 ligand levels in a murine model of RCC. Moreover, the antitumor effect of systemic IL-2 was CXCR3-dependent, as IL-2 failed to inhibit tumor growth and angiogenesis in CXCR3−/− mice. We hypothesized that the immunotherapeutic effect of the CXCR3/CXCR3 ligand biological axis could be optimized by first priming with systemic IL-2 to induce CXCR3 expression on circulating mononuclear cells followed by enhancing the intratumor CXCR3 ligand levels to establish optimal CXCR3-dependent chemotactic gradient. We found that combined systemic IL-2 with an intratumor CXCR3 ligand (CXCL9) lead to significantly greater reduction in tumor growth and angiogenesis, increased tumor necrosis, and increased intratumor infiltration of CXCR3+ mononuclear cells, as compared with either IL-2 or CXCL9 alone. The enhanced antitumor effect of the combined strategy was associated with a more optimized CXCR3-dependent chemotactic gradient and increased tumor-specific immune response. These data suggest that the combined strategy of systemic IL-2 with intratumor CXCR3 ligand is more efficacious than either strategy alone for reducing tumor-associated angiogenesis and augmenting tumor-associated immunity, the concept of immunoangiostasis.
Protein Vaccines Induce Uncommitted IL-2-Secreting Human and Mouse CD4 T Cells, Whereas Infections Induce More IFN-γ-Secreting Cells
Authors: Divekar Anagha A. , Zaiss Dietmar M. W. , Lee F. Eun-Hyung , Liu Dacheng , Topham David J. , Sijts Alice J. A. M. , Mosmann Tim R.
Published: April 2014

Mouse and human CD4 T cells primed during an immune response may differentiate into effector phenotypes such as Th1 (secreting IFN-γ) or Th2 (secreting IL-4) that mediate effective immunity against different classes of pathogen. However, primed CD4 T cells can also remain uncommitted, secreting IL-2 and chemokines, but not IFN-γ or IL-4. We now show that human CD4 T cells primed by protein vaccines mostly secreted IL-2, but not IFN-γ, whereas in the same individuals most CD4 T cells initially primed by infection with live pathogens secreted IFN-γ. We further demonstrate that many tetanus-specific IL-2+IFN-γ− cells are uncommitted and that a single IL-2+IFN-γ− cell can differentiate into Th1 or Th2 phenotypes following in vitro stimulation under appropriate polarizing conditions. In contrast, influenza-specific IL-2+IFN-γ− CD4 cells maintained a Th1-like phenotype even under Th2-polarizing conditions. Similarly, adoptively transferred OTII transgenic mouse T cells secreted mainly IL-2 after priming with OVA in alum, but were biased toward IFN-γ secretion when primed with the same OVA peptide presented as a pathogen Ag during live infection. Thus, protein subunit vaccines may prime a unique subset of differentiated, but uncommitted CD4 T cells that lack some of the functional properties of committed effectors induced by infection. This has implications for the design of more effective vaccines against pathogens requiring strong CD4 effector T cell responses.
Enhanced FTY720-Mediated Lymphocyte Homing Requires Gαi Signaling and Depends on β2 and β7 Integrin
Authors: Pabst Oliver , Herbrand Heike , Willenzon Stefanie , Worbs Tim , Schippers Angela , Müller Werner , Bernhardt Günter , Förster Reinhold
Published: April 2014

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of β2 and β7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either β2 or β7 integrin but not of β2-deficient lymphocytes, which in addition were blocked by anti-β7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Gαi signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.
Splenic Dendritic Cells Induced by Oral Antigen Administration Are Important for the Transfer of Oral Tolerance in an Experimental Model of Asthma
Authors: Nagatani Katsuya , Dohi Makoto , To Yasuo , Tanaka Ryoichi , Okunishi Katsuhide , Nakagome Kazuyuki , Sagawa Kayo , Tanno Yudo , Komagata Yoshinori , Yamamoto Kazuhiko
Published: April 2014

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.
IL-21 Down-Regulates NKG2D/DAP10 Expression on Human NK and CD8+ T Cells
Authors: Burgess Steven J. , Marusina Alina I. , Pathmanathan Ishani , Borrego Francisco , Coligan John E.
Published: April 2014

IL-21 is a recently described cytokine, produced by activated Th cells, that shares significant homology with members of the IL-2 family of cytokines. IL-21 mediates its biological effects via the IL-21R in conjunction with the common receptor γ-chain that is also shared by members of the IL-2 family. We report that culture of human primary NK and CD8+ T cells with IL-21 in combination with IL-2 results in significant reduction of the cell surface expression of NKG2D, compared with that in cells treated with IL-2 alone. The reduced expression of NKG2D after IL-21 culture had functional consequences for NK cell function, as assessed by NKG2D-mediated redirected lysis assays and degranulation assays, compared with NK cells treated with IL-2 alone. IL-21-mediated NKG2D down-regulation in human NK cells correlated with a marked reduction of DNAX-activating protein of 10 kDa (DAP10) transcription in cells treated with IL-2 in combination with IL-21 compared with cells stimulated with only IL-2. This was attributed to a dramatic reduction in DAP10 promoter activity, as assessed by a DAP10 luciferase reporter construct. In contrast to NKG2D expression, IL-21 was able to induce the expression of the NK activation receptors NKp30 and 2B4 as well as the costimulatory receptor CD28 on CD8+ T cells. These data indicate that IL-21 is able to channel NK and CD8+ T cell function by altering the expression pattern of activation/costimulatory receptors.
CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement
Authors: Stone Jennifer D. , Stern Lawrence J.
Published: April 2014

T cell activation is initiated by recognition of antigenic peptide presented in complex with MHC molecules on the surface of APCs. The mechanism by which this recognition occurs is still unclear, and many models exist in the literature. CD4 T cells have been shown to respond to soluble oligomers of activating class II MHC-peptide complexes, but not to soluble monomers. In determining the reactivity of CD8 T cells to soluble activating class I MHC-peptide complexes, a complicating phenomenon had been observed whereby peptide from soluble complexes was loaded onto cell surface MHCs on the T cells and re-presented to other T cells, clouding the true valency requirement for activation. This study uses soluble allogeneic class I MHC-peptide monomers and oligomers to stimulate murine CD8 T cells without the possible complication of peptide re-presentation. The results show that MHC class I monomers bind to, but do not activate, CD8 T cells whether the cells are in solution or adhered to a surface. Monomeric MHC class I binding can antagonize the stimulation triggered by soluble oligomers, a phenomenon also observed for CD4 T cells. Dimeric engagement is necessary and sufficient to stimulate downstream activation processes including TCR down-regulation, Zap70 phosphorylation, and CD25 and CD69 up-regulation, even in T cells that do not express the MHC coreceptor CD8. Thus, the valency dependence of the response of CD8 T cells to soluble MHC-peptide reagents is the same as previously observed for CD4 T cells.
Expansion of Functionally Immature Transitional B Cells Is Associated with Human-Immunodeficient States Characterized by Impaired Humoral Immunity
Authors: Cuss Amanda K. , Avery Danielle T. , Cannons Jennifer L. , Yu Li Jun , Nichols Kim E. , Shaw Peter J. , Tangye Stuart G.
Published: April 2014

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency associated with a marked reduction in circulating memory B cells. Our investigation of the B cell compartment of XLP patients revealed an increase in the frequency of a population of B cells distinct from those previously defined. This population displayed increased expression of CD10, CD24, and CD38, indicating that it could consist of circulating immature/transitional B cells. Supporting this possibility, CD10+CD24highCD38high B cells displayed other immature characteristics, including unmutated Ig V genes and elevated levels of surface IgM; they also lacked expression of Bcl-2 and a panel of activation molecules. The capacity of CD24highCD38high B cells to proliferate, secrete Ig, and migrate in vitro was greatly reduced compared with mature B cell populations. Moreover, CD24highCD38high B cells were increased in the peripheral blood of neonates, patients with common variable immunodeficiency, and patients recovering from hemopoietic stem cell transplant. Thus, an expansion of functionally immature B cells may contribute to the humoral immunodeficient state that is characteristic of neonates, as well as patients with XLP or common variable immunodeficiency, and those recovering from a stem cell transplant. Further investigation of transitional B cells will improve our understanding of human B cell development and how alterations to this process may precipitate immunodeficiency or autoimmunity.
CD27 Dissects Mature NK Cells into Two Subsets with Distinct Responsiveness and Migratory Capacity
Authors: Hayakawa Yoshihiro , Smyth Mark J.
Published: April 2014

Lineage differentiation and the formation of heterogeneous mature subsets are crucial for immune cells to maintain a breadth of responsiveness to pathogens while controlling reactivity to self. In this study, we report that CD27 is a key marker of the NK cell lineage, dissecting the mature Mac-1high NK cell pool into two functionally distinct subsets. The CD27low NK cell subset possesses a higher threshold to stimulation and appears to be tightly regulated by the expression of NK cell inhibitory receptors. Comparatively, the CD27high NK cell subset displays a greater effector function, exhibits a distinct tissue distribution and responsiveness to chemokines, and interacts productively with dendritic cells. Importantly, we have verified that CD27high and CD27low subsets with distinct cell surface phenotypes also exist in human peripheral blood. These findings clearly reclassify mature NK cells into two distinct subsets and begin to discern their specific role in immune responses.
Therapeutic Vaccination of Active Arthritis with a Glycosylated Collagen Type II Peptide in Complex with MHC Class II Molecules
Authors: Dzhambazov Balik , Nandakumar Kutty Selva , Kihlberg Jan , Fugger Lars , Holmdahl Rikard , Vestberg Mikael
Published: April 2014

In both collagen-induced arthritis (CIA) and rheumatoid arthritis, T cells recognize a galactosylated peptide from type II collagen (CII). In this study, we demonstrate that the CII259–273 peptide, galactosylated at lysine 264, in complex with Aq molecules prevented development of CIA in mice and ameliorated chronic relapsing disease. In contrast, nonglycosylated CII259–273/Aq complexes had no such effect. CIA dependent on other MHC class II molecules (Ar/Er) was also down-regulated, indicating a bystander vaccination effect. T cells could transfer the amelioration of CIA, showing that the protection is an active process. Thus, a complex between MHC class II molecules and a posttranslationally modified peptide offers a new possibility for treatment of chronically active autoimmune inflammation such as rheumatoid arthritis.
Pregnancy-Associated Exosomes and Their Modulation of T Cell Signaling
Authors: Taylor Douglas D. , Akyol Sibel , Gercel-Taylor Cicek
Published: April 2014

Exosome release by viable cells is a feature of activated cell types, including tumors, fetal cells, and cells of the immune system. Exosomes critically regulate immune activation, by mediating activation-induced cell death. Fetal cells may mimic these events to selectively delete reactive lymphocytes. In this study the presence and composition of placenta-derived exosomes are demonstrated in the maternal circulation along with their consequences on T cell activation markers. For all pregnant patients, exosomes were isolated from sera obtained between 28 and 30 wk gestation. For pregnant women, subsequently delivering at term, circulating levels of placental exosomes were 1.8 times greater than those delivering preterm (p < 0.0001). Exosomes isolated from pregnancies subsequently delivering at term expressed significantly higher levels of biologically active components, including Fas ligand (FasL) and HLA-DR, than those from pregnancies delivering preterm. Standardizing for protein concentrations, exosomes from term-delivering pregnancies exhibited greater suppression of CD3-ζ and JAK3 than those delivering preterm. The suppression of CD3-ζ and JAK3 correlated with exosome expression levels of FasL (r2 = 0.92 and r2 = 0.938, respectively). Fractionation of exosomes from term-delivering pregnancies by continuously eluting electrophoresis indicated that intact 42kD FasL and an unidentified 24-kDa protein were associated with CD3-ζ suppression. Our results demonstrated that exosomes from pregnancies ultimately delivering at term are present at significantly greater concentrations than those from pregnancies delivering preterm; however, exosomes from term-delivering pregnancies also exhibit significantly greater suppression of CD3-ζ and JAK3.
Human αβ and γδ Thymocyte Development: TCR Gene Rearrangements, Intracellular TCRβ Expression, and γδ Developmental Potential—Differences between Men and Mice
Authors: Joachims Michelle L. , Chain Jennifer L. , Hooker Scott W. , Knott-Craig Christopher J. , Thompson Linda F.
Published: April 2014

To evaluate the role of the TCR in the αβ/γδ lineage choice during human thymocyte development, molecular analyses of the TCRβ locus in γδ cells and the TCRγ and δ loci in αβ cells were undertaken. TCRβ variable gene segments remained largely in germline configuration in γδ cells, indicating that commitment to the γδ lineage occurred before complete TCRβ rearrangements in most cases. The few TCRβ rearrangements detected were primarily out-of-frame, suggesting that productive TCRβ rearrangements diverted cells away from the γδ lineage. In contrast, in αβ cells, the TCRγ locus was almost completely rearranged with a random productivity profile; the TCRδ locus contained primarily nonproductive rearrangements. Productive γ rearrangements were, however, depleted compared with preselected cells. Productive TCRγ and δ rearrangements rarely occurred in the same cell, suggesting that αβ cells developed from cells unable to produce a functional γδ TCR. Intracellular TCRβ expression correlated with the up-regulation of CD4 and concomitant down-regulation of CD34, and plateaued at the early double positive stage. Surprisingly, however, some early double positive thymocytes retained γδ potential in culture. We present a model for human thymopoiesis which includes γδ development as a default pathway, an instructional role for the TCR in the αβ/γδ lineage choice, and a prolonged developmental window for β selection and γδ lineage commitment. Aspects that differ from the mouse are the status of TCR gene rearrangements at the nonexpressed loci, the timing of β selection, and maintenance of γδ potential through the early double positive stage of development.
Responsiveness of Naive CD4 T Cells to Polarizing Cytokine Determines the Ratio of Th1 and Th2 Cell Differentiation
Authors: Mikhalkevich Natallia , Becknell Brian , Caligiuri Michael A. , Bates Michael D. , Harvey Richard , Zheng Wei-ping
Published: April 2014

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4Rα. The down-regulation of IL-4Rα diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4Rα with Abs had the same effect in an Ab dose-dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4Rα to favor Th2 cell differentiation. Thus, the IL-4Rα level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation.
TLR Agonists Abrogate Costimulation Blockade-Induced Prolongation of Skin Allografts
Authors: Thornley Thomas B. , Brehm Michael A. , Markees Thomas G. , Shultz Leonard D. , Mordes John P. , Welsh Raymond M. , Rossini Aldo A. , Greiner Dale L.
Published: April 2014

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used “synchimeric” mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2−/− and B6.IL-12R−/− mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8α−/− and TLR4−/− mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.
Tec Kinases Itk and Rlk Are Required for CD8+ T Cell Responses to Virus Infection Independent of Their Role in CD4+ T Cell Help
Authors: Atherly Luana O. , Brehm Michael A. , Welsh Raymond M. , Berg Leslie J.
Published: April 2014

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4+ T cells, particularly in the phosphorylation of phospholipase C-γ1 after TCR activation; furthermore, both Itk and Rlk are important in CD4+ T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8+ T cell signaling and function. Using Itk−/− and Itk−/−Rlk−/− mice, we examined the roles of these Tec family kinases in CD8+ T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-γ1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8+ T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4+ T cell help, thereby substantiating the important role of Tec kinases in CD8+ T cell signaling.
CD4+CD25+ T Regulatory Cells Suppress NK Cell-Mediated Immunotherapy of Cancer
Authors: Smyth Mark J. , Teng Michele W. L. , Swann Jeremy , Kyparissoudis Konstantinos , Godfrey Dale I. , Hayakawa Yoshihiro
Published: April 2014

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-β-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.
Brain-Derived Heat Shock Protein 70-Peptide Complexes Induce NK Cell-Dependent Tolerance to Experimental Autoimmune Encephalomyelitis
Authors: Galazka Grazyna , Stasiolek Mariusz , Walczak Agata , Jurewicz Anna , Zylicz Alicja , Brosnan Celia F. , Raine Cedric S. , Selmaj Krzysztof W.
Published: April 2014

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139–151 (PLP139–151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139–151 that correlated with elevated IFN-γ and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139–151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.