bluuebunny/crossref_metadata_2025 huggingface dataset:
Editing Autoreactive TCR Enables Efficient Positive Selection
Authors: Santori Fabio R. , Arsov Ivica , Lili Mirjana , Vukmanović Stanislav
Published: April 2014

Allelic exclusion is inefficient at the TCRα locus, allowing a sizeable portion of T cells to carry two functional TCRs. The potential danger of dual TCR expression is a rescue of autoreactive TCRs during selection in the thymus and subsequent development of autoimmunity. In this study, we examine the reason(s) for replacing an autoreactive TCR and for allowing the survival of cells carrying two TCRs. We compared development of TCR transgenic CD4+CD8− thymocytes in the presence or absence of MHC class II autoantigen that does not induce deletion of thymocytes. Contrary to the expected negative effect of the presence of autoantigen, ∼100% more CD4+CD8− thymocytes were found in the presence of MHC class II autoantigen than in the neutral background. A further increase in the strength of autoantigenic signal via expression of a human CD4 transgene led to an additional increase in the numbers of CD4+CD8− thymocytes. Thus, editing autoreactive TCR results in more efficient positive selection, and this may be both a reason and a reward for risking autoimmunity.
The Unique Antigen Receptor Signaling Phenotype of B-1 Cells Is Influenced by Locale but Induced by Antigen
Authors: Chumley Michael J. , Dal Porto Joseph M. , Cambier John C.
Published: April 2014

Normal animals contain an autoreactive B lymphocyte subset, the B-1 subset, which is controlled by undefined mechanisms to prevent autoimmunity. Using a VH11Vκ9 Ig transgenic mouse, with a specificity prototypic of the subset, we have explored conditions responsible for the previously reported Ag hyporesponsiveness of these cells. We report that peritoneal VH11Vκ9 B cells exhibit typical B-1 behavior with high basal intracellular free Ca2+ and negligible receptor-mediated calcium mobilization. However, splenic B cells from this mouse, while phenotypically similar to their peritoneal counterparts, including expression of CD5, mount robust B-2-like responses to Ag as measured by calcium influx and altered tyrosine phosphorylation responses. When these splenic cells are adoptively transferred to the peritoneal cavity and encounter their cognate self-Ag, they acquire a B-1 signaling phenotype. The ensuing hyporesponsiveness is characterized by increases in both basal intracellular calcium and resting tyrosyl phosphorylation levels and is highlighted by a marked abrogation of B cell receptor-mediated calcium mobilization. Thus, we show that self-Ag recognition in specific microenvironments such as the peritoneum, and we would propose other privileged sites, confers a unique form of anergy on activated B cells. This may explain how autoreactive B-1 cells can exist while autoimmunity is avoided.
Role of B7 Costimulatory Molecules in the Adjuvant Activity of the Heat-Labile Enterotoxin ofEscherichia coli
Authors: Martin Michael , Sharpe Arlene , Clements John D. , Michalek Suzanne M.
Published: April 2014

Much interest has been directed at understanding the adjuvant properties of the heat-labile enterotoxin of Escherichia coli (LT). In this study, we have assessed how LT compared with the nonenzymatic mutant LT (E112K) affect the level of B7-1 and B7-2 expression on APCs, and we determined how these costimulatory molecules influence their adjuvant properties. Analysis of B7-1 and B7-2 expression on B cells revealed that LT enhanced B7-2 but not B7-1, while LT (E112K) had no effect on the expression of either costimulatory molecule. Treatment of macrophage or dendritic cells with LT resulted in a predominant enhancement of B7-2, while LT (E112K) induced mainly B7-1 expression. Analysis of LT- and LT (E112K)-treated B cells, macrophage, and dendritic cells also revealed significant differences in their ability to enhance anti-CD3-stimulated CD4+ T cell proliferative responses via B7-1 and B7-2. Furthermore, the ability of LT to enhance both Ab and CD4+ T cell responses to a coadministered Ag was severely abrogated in B7-2- but not B7-1-deficient mice. In contrast, the in vivo adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4+ T cell responses, while B7-1 appeared to be the predominant B7 molecule involved in the ability of LT (E112K) to augment Ab responses to a coadministered Ag. These findings demonstrate distinct differences in the ability of LT and LT (E112K) to enhance B7-1 and B7-2 on APC, as well as a dependence upon these costimulatory molecules for their adjuvant properties.
Opposing Effects of IL-2 in Tumor Immunotherapy: Promoting CD8 T Cell Growth and Inducing Apoptosis
Authors: Shrikant Protul , Mescher Matthew F.
Published: April 2014

Tumors often induce specific CTL responses, but these are usually ineffective at eliminating the growing tumor. The T cell growth factor IL-2 has potential for expanding and prolonging CTL responses, and there is considerable interest in using this cytokine in combination with other immunotherapeutic agents that target T cell responses. Using adoptive transfer of OT-I CD8 T cells specific for OVA257–264 peptide, and E.G7 tumor cells transfected with OVA, we have examined the effects of IL-2 on the generation and maintenance of a CTL response to the tumor. Administration of IL-2 during the initial phase of the response, clonal expansion, and development of effector function, had no effect on the number of CTL generated or the control of tumor growth. In contrast, a short 2-day time course of low-dose IL-2 at the peak of clonal expansion or at later times resulted in prolonged and expanded responses by the OT-I CTL, with concomitant decrease in tumor load and extension of survival. However, when IL-2 administration was more prolonged, as is often the case in clinical trials, the therapeutic benefit was lost due to elimination of the tumor-specific CTL, at least in part through induction of apoptosis. These results demonstrate that use of IL-2 for tumor immunotherapy is very much a double-edged sword and strongly suggest that more limited time and dose regimens may substantially improve its clinical efficacy when it is used in conjunction with approaches that target CTL responses.
Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Express Functional IL-15 Receptor Complex: Endogenous IL-15 in Autocrine Fashion Enhances Cell Proliferation and Expression of Bcl-xL and Bcl-2
Authors: Kurowska Mariola , Rudnicka Weronika , Kontny Ewa , Janicka Iwona , Chorazy Magdalena , Kowalczewski Jacek , Ziółkowska Maria , Ferrari-Lacraz Sylvie , Strom Terry B. , Maśliński Włodzimierz
Published: April 2014

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Rα at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rβ and common γ-chains. Interestingly, TNF-α or IL-1β triggered significant elevation of IL-15Rα chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-xL expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-xL. To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcγ2a protein exerting properties of specific antagonist to the IL-15Rα chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-xL, and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.
L-Selectin Serves as an E-Selectin Ligand on Cultured Human T Lymphoblasts
Authors: Jutila Mark A. , Kurk Sandy , Jackiw Larrisa , Knibbs Randall N. , Stoolman Lloyd M.
Published: April 2014

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by ∼60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, ∼60–70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.
Expression and Function of Transgenic HLA-DQ Molecules and Lymphocyte Development in Mice Lacking Invariant Chain
Authors: Rajagopalan Govindarajan , Smart Michele K. , Krco Christopher J. , David Chella S.
Published: April 2014

Invariant chain (Ii) is a non-MHC-encoded molecule, which plays an accessory role in the proper assembly/expression of functional MHC class II molecules and there by plays an important role in Ag processing/presentation. The phenotype of mice lacking Ii depends on the allotype of the MHC class II molecule. In some mice strains, Ii deficiency results in reduction in expression of class II molecules accompanied by defective CD4+ T cell development. Responses to conventional Ags/superantigens are also compromised. In this study, we describe for the first time the functionality of human class II molecules, HLA-DQ6 and HLA-DQ8, in transgenic mice lacking Ii. HLA transgenic Ii−/− mice expressed very low levels of surface DQ6 and DQ8 accompanied by severe reduction in CD4+ T cells both in the thymus and periphery. In vitro proliferation and cytokine production to an exogenous superantigen, staphylococcal enterotoxin B (SEB) was diminished in HLA-transgenic Ii−/− mice. However, SEB-induced in vivo expansion of CD8+ T cells expressing TCR Vβ8 family in DQ8.Ii−/− mice was comparable with that of DQ8.Ii+/+ mice. Systemic IFN-γ production following in vivo challenge with SEB was reduced in DQ8.Ii−/− mice and were also protected from SEB-induced toxic shock. Although the T cell response to a known peptide Ag was diminished in DQ8.Ii−/− mice, DQ8.Ii−/− APCs were capable of presenting that peptide to primed T cells from wild-type DQ8 mice as well as to a specific T cell hybridoma. Differentiation of mature B cells was also affected to a certain extent in DQ8.Ii−/− mice.
CD30 Expression Identifies the Predominant Proliferating T Lymphocyte Population in Human Alloimmune Responses
Authors: Chan Keith W. , Hopke Corwyn D. , Krams Sheri M. , Martinez Olivia M.
Published: April 2014

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30+ T cells elicited with allogeneic APC are a major source of IFN-γ and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30+ T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30+ T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30+ cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.
4-1BB-Specific Monoclonal Antibody Promotes the Generation of Tumor-Specific Immune Responses by Direct Activation of CD8 T Cells in a CD40-Dependent Manner
Authors: Miller Robert E. , Jones Jon , Le Tiep , Whitmore James , Boiani Norman , Gliniak Brian , Lynch David H.
Published: April 2014

4-1BB (CD137) is a member of the TNFR superfamily (TNFRSF9). T cell expression of 4-1BB is restricted to activated cells, and cross-linking has been shown to deliver a costimulatory signal. Here we have shown that treatment of tumor-bearing mice with agonistic 4-1BB-specific Abs can lead to T cell-mediated tumor rejection. In vivo mAb depletion experiments demonstrated that this rejection requires CD8+ cells but not CD4+ or NK cells. Both IFN-γ- and CD40-mediated signals were also required, because no benefit was observed on treatment with 4-1BB mAb in mice in which the genes for these molecules had been knocked out. Interestingly, 4-1BB-mediated stimulation of immune responses in CD40L−/− mice is effective (although at a reduced level), and may suggest the existence of an alternative ligand for CD40. Additional experiments in IL-15−/− mice indicate that IL-15 is not required for either the generation of the primary tumor-specific immune response or the maintenance of the memory immune response. In contrast, the presence of CD4 cells during the primary immune response appears to play a significant role in the maintenance of effective antitumor memory. Finally, in mice in which the number of dendritic cells had been expanded by Fms-like tyrosine kinase3 ligand treatment, the antitumor effects of 4-1BB ligation were enhanced.
The Th1-Specific Costimulatory Molecule, M150, Is a Posttranslational Isoform of Lysosome-Associated Membrane Protein-1
Authors: Prasad Durbaka V. R. , Parekh Vrajesh V. , Joshi Bimba N. , Banerjee Pinaki P. , Parab Pradeep B. , Chattopadhyay Samit , Kumar Anil , Mishra Gyan C.
Published: April 2014

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4+ T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4+ T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.
FcεRI Induces the Tryptophan Degradation Pathway Involved in Regulating T Cell Responses
Authors: von Bubnoff Dagmar , Matz Heike , Frahnert Christine , Rao Marie Luise , Hanau Daniel , de la Salle Henri , Bieber Thomas
Published: April 2014

FcεRI is suspected to play a pivotal role in the pathophysiology of atopic disorders such as atopic dermatitis. In search for genes differentially regulated by FcεRI on APCs, a differential cDNA bank of receptor-stimulated and unstimulated monocytes was established. By means of suppression subtractive hybridization, we identified kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase (IDO) to be overexpressed in FcεRI-activated monocytes. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. We show that cross-linking of FcεRI on monocytes results in low tryptophan concentrations associated with impaired T cell stimulatory capacity. Importantly, T cell suppression could be prevented by the addition of tryptophan or inhibition of IDO. Moreover, stimulation of T cells by FcεRI-activated monocytes was increased compared with T cell stimulation by nonactivated monocytes if exogenous supply of tryptophan was available. We speculate that the expression of IDO by FcεRI+ APCs in vivo allows these cells to regulate T cell responses in atopic disorders by inhibiting or stimulating T cell proliferation, depending on the metabolic environment.
A Reassessment of the Effect of Activated Notch1 on CD4 and CD8 T Cell Development
Authors: Fowlkes B. J. , Robey Ellen A.
Published: April 2014

The Notch signaling pathway plays an important role in the early steps of T cell development and in the generation of T cell tumors, but its role in the CD4 vs CD8 lineage decision is controversial. Notch1 is not essential for CD4 or CD8 T cell development; however, there are suggestions that multiple Notch family members may act in a redundant fashion during thymic development. In theory, expressing a constitutively activated form of Notch in CD4+CD8+ thymocytes could provide clues about the normal role of Notch in developing CD4 and CD8 T cells. Unfortunately, two different studies of transgenic mice expressing activated forms of Notch1 (Notch1IC) led to conflicting conclusions. In this study, we re-examine the effect of the two Notch1IC transgenes on thymocyte development. We find that both Notch1IC transgenic lines display a decrease in CD4 single positive (SP) thymocytes and a corresponding increase in CD8 SP thymocytes. The enhanced development of CD8 SP thymocytes is dependent on either class I or II MHC. Thus, data from two different Notch1IC transgenic lines indicate that Notch activity promotes CD8 and inhibits CD4 SP development. We suggest that the discrepancies in previous reports of Notch1IC transgenic mice are due to differences in the propensity of the two different transgenic lines to develop tumors.
The Poststimulation Program of CD4 Versus CD8 T Cells (Death Versus Activation-Induced Nonresponsiveness)
Authors: Tham Ee Loon , Mescher Matthew F.
Published: April 2014

Both CD8 and CD4 T cells undergo autocrine IL-2-induced proliferation and clonal expansion following stimulation with Ag and costimulation. The CD8 T cell response is transient because the cells rapidly become activation-induced nonresponsive (AINR) and exhibit split anergy. In these cells, the capacity for IL-2 production is lost, but TCR-mediated IFN-γ production and cytotoxicity are maintained. At this point, the CTL become dependent on IL-2 provided by CD4 Th cells for continued expansion. If IL-2 is available to support expansion for a brief period, AINR is reversed and the cells regain the ability to produce IL-2. In this study, we show that CD4 T cells do not become AINR, but instead are rendered susceptible to Fas-mediated activation-induced cell death following stimulation through TCR and CD28. Using z-VAD-fmk or anti-Fas ligand mAb to inhibit cell death, we demonstrate that previously activated CD4 T cells retain the ability to up-regulate c-Jun N-terminal kinase activity and IL-2 mRNA levels upon TCR engagement and no longer require costimulation. This rewiring of signaling pathways is similar to that seen following reversal of AINR in CD8 T cells. Thus, CD8 and CD4 T cells appear to use distinct mechanisms, AINR and activation-induced cell death, respectively, to limit excessive clonal expansion following a productive response, while permitting important effector functions to be expressed.
Antigen-Driven Oligoclonal Expansion of Tumor-Infiltrating B Cells in Infiltrating Ductal Carcinoma of the Breast
Authors: Coronella Julia A. , Spier Catherine , Welch Matthew , Trevor Katrina T. , Stopeck Alison T. , Villar Hugo , Hersh Evan M.
Published: April 2014

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20+ B lymphocyte and CD3+ T lymphocyte zones with interdigitating CD21+ follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45–68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.
Thymocyte Apoptosis Induced by T Cell Activation Is Mediated by Glucocorticoids In Vivo
Authors: Brewer Judson A. , Kanagawa Osami , Sleckman Barry P. , Muglia Louis J.
Published: April 2014

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3ε Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3ε-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.
Selective Adherence of IgA to Murine Peyer’s Patch M Cells: Evidence for a Novel IgA Receptor
Authors: Mantis Nicholas J. , Cheung Marsha C. , Chintalacharuvu Koteswara R. , Rey Jacques , Corthésy Blaise , Neutra Marian R.
Published: April 2014

M cells represent the primary route by which mucosal Ags are transported across the intestinal epithelium and delivered to underlying gut-associated lymphoid tissues. In rodents and rabbits, Peyer’s patch M cells selectively bind and endocytose secretory IgA (SIgA) Abs. Neither the nature of the M cell IgR nor the domains of SIgA involved in this interaction are known. Using a mouse ligated ileal loop assay, we found that monoclonal IgA Abs with or without secretory component, but not IgG or IgM Abs, bound to the apical surfaces of Peyer’s patch M cells, indicating that the receptor is specific for the IgA isotype. Human serum IgA and colostral SIgA also bound to mouse M cells. The asialoglycoprotein receptor or other lectin-like receptors were not detected on the apical surfaces of M cells. We used recombinant human IgA1 and human IgA2 Abs and domain swapped IgA/IgG chimeras to determine that both domains Cα1 and Cα2 are required for IgA adherence to mouse Peyer’s patch M cells. This distinguishes the M cell IgA receptor from CD89 (FcαI), which binds domains Cα2-Cα3. Finally, we observed by immunofluorescence microscopy that some M cells in the human ileum are coated with IgA. Together these data suggest that mouse, and possibly human, M cells express an IgA-specific receptor on their apical surfaces that mediates the transepithelial transport of SIgA from the intestinal lumen to underlying gut-associated organized lymphoid tissues.
Re-establishing Peripheral Tolerance in the Absence of CTLA-4: Complementation by Wild-Type T Cells Points to an Indirect Role for CTLA-4
Authors: Tivol Elizabeth A. , Gorski Jack
Published: April 2014

CTLA-4 plays an important role in the down-regulation of activated T cells and in the establishment of peripheral tolerance. It has been hypothesized that CTLA-4 on the cell surface signals directly into T cells during primary immune responses, resulting in intrinsic T cell down-regulation. It is not known, however, whether CTLA-4 directly inhibits the less intense activating signals received by autoreactive T cells in the periphery. We investigated whether CTLA-4 acts intrinsically upon self-reactive cells in vivo, or whether it inhibits autoreactive cells indirectly, in a non-cell autonomous manner. The adoptive transfer of CTLA-4-deficient splenocytes or Thy 1+ cells into recombinase-activating gene 2-deficient mice resulted in fatal inflammation and tissue destruction similar to that seen in CTLA-4-deficient mice. When an equivalent number of splenocytes or Thy 1+ cells from wild-type animals was transferred with the CTLA-4-deficient cells, recipient mice survived indefinitely. Since CTLA-4 was absent in the T cells responsible for the inflammatory phenotype, the down-regulation of these autoreactive cells must have been facilitated indirectly by wild-type Thy 1+ cells. In addition, a rapid reduction in the ratio of CTLA-4-deficient to wild-type cells was observed. We propose two possible indirect mechanisms by which CTLA-4 may function in the establishment and maintenance of peripheral tolerance.
Differential Expression of the Inhibitory IgG Fc Receptor FcγRIIB on Germinal Center Cells: Implications for Selection of High-Affinity B Cells
Authors: Rao Sambasiva P. , Vora Kalpit A. , Manser Tim
Published: April 2014

The murine low-affinity receptor for IgG, FcγRIIB, mediates inhibition of B cell receptor-triggered events in primary B cells. We investigated the expression of FcγRIIB on germinal center (GC) cells to better understand its role in memory B cell development. Immunohistological analyses demonstrated differential regulation of FcγRIIB on GC cells. Its levels are markedly down-regulated on GC B cells and up-regulated on follicular dendritic cells (FDC) at all times during the GC response. Analyses of surface expression of FcγRIIB by flow cytometry and FcγRIIB mRNA levels by RT-PCR analysis confirmed that this FcR is down-regulated in GC B cells. In mice lacking FcγRIIB, the development of the secondary FDC reticulum in GCs is substantially delayed, although the overall kinetics of the GC response are unaltered. These findings have direct implications for models proposed to account for the selection of high-affinity B cells in the GC and suggest a role for FcγRIIB in promoting the maturation of the FDC reticulum.
Sonic Hedgehog Promotes Cell Cycle Progression in Activated Peripheral CD4+ T Lymphocytes
Authors: Lowrey Jacqueline A. , Stewart Gareth A. , Lindey Susannah , Hoyne Gerard F. , Dallman Margaret J. , Howie Sarah E. M. , Lamb Jonathan R.
Published: April 2014

Sonic hedgehog (Shh) signaling is important in the growth and differentiation of many cell types and recently has been reported to play a role in T cell development in the thymus. This prompted us to investigate whether or not Shh contributes to the clonal expansion of peripheral CD4+ T cells. In this study, we demonstrate that Shh and other components of the signaling pathway patched, smoothened, and Gli1 (glioma-associated oncogene) are expressed in peripheral CD4+ T cells. The addition of the biologically active amino-terminal Shh peptide had no effect on resting CD4+ T cells, but significantly enhanced proliferation of anti-CD3/28 Ab-activated CD4+ T cells. This was not due to antiapoptotic effects, but by promoting entry of T cells into the S-G2 proliferative phase of the cell cycle. Neutralizing anti-Shh Ab reduced T cell proliferation by inhibiting cell transition into the S-G2 phase, suggesting that endogenously produced Shh plays a physiological role in the clonal expansion of T cells. Furthermore, we have observed a significant up-regulation of Shh and Gli1 (glioma-associated oncogene) mRNA in activated CD4+ T cells with or without addition of exogenous Shh, which corresponds with maximal CD4+ T cell proliferation, whereas bcl-2 was only up-regulated in activated cells in the presence of Shh. Our findings suggest that endogenously produced Shh may play a role in sustaining normal CD4+ T cell proliferation and exogenously added Shh enhances this response.
The Human IL-3 Locus Is Regulated Cooperatively by Two NFAT-Dependent Enhancers That Have Distinct Tissue-Specific Activities
Authors: Hawwari Abbas , Burrows Joanna , Vadas Mathew A. , Cockerill Peter N.
Published: April 2014

The human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the −4.5-kb enhancer cooperated with the −14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at −4.1 kb in T cells, mast cells, and CD34+ myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.
T Cell Recognition of an Engineered MHC Class I Molecule: Implications for Peptide-Independent Alloreactivity
Authors: Janković Vladimir , Remus Kristin , Molano Alberto , Nikolich-Žugich Janko
Published: April 2014

Previously, we described H-2KbW9 (KbW9), an engineered variant of the murine MHC class I molecule H-2Kb (Kb), devoid of the central anchor (“C”) pocket owing to a point mutation on the floor of the peptide binding site; this substitution drastically altered selection of bound peptides, such that the peptide repertoires of Kb and KbW9 are largely nonoverlapping in vivo. On the basis of these observations, we used KbW9 and Kb to revisit the role of peptides in alloreactive T cell recognition. We first compared Ab and TCR recognition of KbW9 and Kb. Six of six Kb-specific mAbs, directed against different parts of the molecule, recognized KbW9 well, albeit at different levels than Kb. Furthermore, KbW9 readily served as a restriction element for a peptide-specific syngeneic CTL response. Therefore, KbW9 mutation did not result in gross distortions of the TCR-interacting surface of class I, which was comparable between Kb and KbW9. Interestingly, when KbW9 was used to stimulate allogeneic T cells, it induced an infrequent CTL population that cross-reacted against Kb and was specific for peptide-independent MHC epitopes. By contrast, Kb-induced alloreactive CTLs recognized Kb in a peptide-specific manner, did not cross-react on KbW9, and were present at much higher frequencies than those induced by KbW9. Thus, induction of rare peptide-independent CTLs depended on unique structural features of KbW9, likely due to the elevated floor of the peptide-binding groove and the consequent protruding position of the peptide. These results shed new light on the relationship between TCR and peptide-MHC complex in peptide-independent allorecognition.
DNA Methylation Changes at Human Th2 Cytokine Genes Coincide with DNase I Hypersensitive Site Formation During CD4+ T Cell Differentiation
Authors: Santangelo Samantha , Cousins David J. , Winkelmann Nicole E. E. , Staynov Dontcho Z.
Published: April 2014

The differentiation of naive CD4+ T lymphocytes into Th1 and Th2 lineages generates either cellular or humoral immune responses. Th2 cells express the cytokines IL-4, -5, and -13, which are implicated in asthma and atopy. Much has been published about the regulation of murine Th2 cytokine expression, but studies in human primary T cells are less common. We have developed a method for differentiating human CD45RA+ (naive) T cells into Th1 and Th2 populations that display distinct cytokine expression profiles. We examined both CpG methylation, using bisulfite DNA modification and sequencing, and chromatin structure around the IL-4 and IL-13 genes before and after human T cell differentiation and in normal human skin fibroblasts. In naive cells, the DNA was predominantly methylated. After Th2 differentiation, DNase I hypersensitive sites (DHS) appeared at IL-4 and IL-13 and CpG demethylation occurred only around the Th2-specific DHS. Both DHS and CpG demethylation coincided with consensus binding sites for the Th2-specific transcription factor GATA-3. Although fibroblasts, like naive and Th1 cells, did not express IL-4 or IL-13, DHS and unmethylated CpG sites that were distinct from the Th2-specific sites were observed, suggesting that chromatin structure in this cluster not only varies in T cells according to IL-4/IL-13 expression but is also tissue specific.
Early Events of TCR Signaling Are Distinct in Human Th1 and Th2 Cells
Authors: Hannier Sigrid , Bitegye Christina , Demotz Stéphane
Published: April 2014

To study the requirements for activation of human Th1 and Th2 cells, soluble peptide/DR1 complexes were prepared from naturally expressed DR1 protein. When immobilized, this material induced T cell activation, as revealed by CD25 up-regulation. Unexpectedly, Th2 cells required a higher density of peptide/DR1 complexes than Th1 cells to initiate CD25 up-regulation. Similar findings were obtained with immobilized or soluble and cross-linked anti-CD3 mAb. In contrast, peptide/DR1 complexes displayed on the surface of nonprofessional APC similarly induced CD25 up-regulation in Th1 and Th2 cells. Signaling events distinguishing human Th1 and Th2 cells following TCR engagement by anti-CD3 mAb were then studied. It was observed that upon TCR triggering, the overall tyrosine phosphorylation profiles were fainter in Th2 than in Th1 clones. Similar results were obtained with Th1- and Th2-polarized polyclonal lines. Varying the dose of anti-CD3 mAb, the kinetics of activation, and coengagement of CD3 and CD28 failed to increase tyrosine phosphorylation in Th2 cells to levels reached in Th1 cells. In contrast, treatment with the tyrosine phosphatase inhibitor phenylarsine oxide resulted in similar tyrosine phosphorylation levels in Th2 and Th1 cells. These findings indicated that Th2 cells had an intrinsically lower TCR-induced tyrosine phosphorylation capacity than Th1 cells, which might be controlled by Th1- and Th2-specific phosphatase profiles. Finally, a weaker association was found between ZAP-70 and CD3ζ in Th2 than in Th1 cells after TCR engagement. Taken together, these results constituted evidence that early events in the TCR signaling cascades are distinct in human Th1 and Th2 cells.
Transcriptional Regulation of the Human Polymeric Ig Receptor Gene: Analysis of Basal Promoter Elements
Authors: Hempen Paula M. , Phillips Kimberly M. , Conway Pamela S. , Sandoval Katrina H. , Schneeman Tracey A. , Wu Hsin-Jung , Kaetzel Charlotte S.
Published: April 2014

Secretory Igs provide the first line of adaptive immune defense against ingested, inhaled, and sexually transmitted pathogens at mucosal surfaces. The polymeric Ig receptor regulates transport of dimeric IgA and pentameric IgM into external secretions. The level of expression of polymeric Ig receptor is controlled to a large extent by transcription of the PIGR gene in mucosal epithelial cells. Here we present a detailed analysis of the promoter of the PIGR gene by transient transfection of luciferase reporter plasmids into cultured cell lines. Comparisons of the human and mouse PIGR promoters in human and mouse intestinal and liver cell lines demonstrated that the human PIGR promoter was 4- to 5-fold more active than the mouse PIGR promoter in all cell types, and that both the human and mouse PIGR promoters were more active in intestinal than in liver cell lines. Targeted deletions of 22-bp segments of the human PIGR promoter revealed that the region from nt −63 to −84 is crucial for basal transcription, and that two upstream regions can act as positive or negative regulators. Point mutations within the region from nt −63 to −84 demonstrated that an E box motif, which binds the basic helix-loop-helix protein upstream stimulatory factor, is required for PIGR promoter activity. Two additional regulatory motifs were identified in the proximal promoter region: a binding site for AP2, and an inverted repeat motif that binds an unidentified protein. These findings suggest that cooperative binding of multiple transcription factors regulates basal activity of the human PIGR promoter.
Repression of AP-1 Function: A Mechanism for the Regulation of Blimp-1 Expression and B Lymphocyte Differentiation by the B Cell Lymphoma-6 Protooncogene
Authors: Vasanwala Farha H. , Kusam Saritha , Toney Lisa M. , Dent Alexander L.
Published: April 2014

The B cell lymphoma-6 (BCL-6) transcriptional repressor protein is an important regulator of B cell differentiation and is strongly implicated in the development of B cell lymphoma. Expression of the Blimp-1 transcription factor, which is critical for promoting B cell differentiation into plasma cells, is repressed by BCL-6. We have investigated the mechanism for how BCL-6 represses Blimp-1 transcription, and have found that BCL-6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. Specifically, BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We found that the zinc-finger region of BCL-6 interacts with c-Jun, JunB, and JunD proteins but does not bind c-Fos or Fra-2 proteins. An estrogen receptor ligand binding domain fusion with the BCL-6 zinc finger domain can act as a estrogen-inducible dominant negative protein and increase AP-1 activity in BCL-6+ cells but not in BCL-6− cells, indicating that endogenous BCL-6 represses AP-1 activity. Additionally, we have confirmed a specific interaction between c-Jun and the zinc finger domain of BCL-6 in vivo using a mammalian two-hybrid assay. Repression of AP-1 function by BCL-6 may be a key mechanism for how BCL-6 regulates gene expression to control inflammation, lymphocyte differentiation, and lymphomagenesis.
Induction of Central Deletional T Cell Tolerance by Gene Therapy
Authors: Kang Eun-Suk , Iacomini John
Published: April 2014

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag Kb were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2Kb resulted in lifelong expression of Kb on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2Kb transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with Kb transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to Kb. Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.
A Novel Functional Class I Lineage in Zebrafish (Danio rerio), Carp (Cyprinus carpio), and Large Barbus (Barbus intermedius) Showing an Unusual Conservation of the Peptide Binding Domains
Authors: Kruiswijk Corine P. , Hermsen Trudi T. , Westphal Adrie H. , Savelkoul Huub F. J. , Stet René J. M.
Published: April 2014

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.
Role That Each NKG2A Immunoreceptor Tyrosine-Based Inhibitory Motif Plays in Mediating the Human CD94/NKG2A Inhibitory Signal
Authors: Kabat Juraj , Borrego Francisco , Brooks Andrew , Coligan John E.
Published: April 2014

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6–11) and membrane-proximal (residues 38–43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcεRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.
Lipid Microdomains Are Required Sites for the Selective Endocytosis and Nuclear Translocation of IFN-γ, Its Receptor Chain IFN-γ Receptor-1, and the Phosphorylation and Nuclear Translocation of STAT1α
Authors: Subramaniam Prem S. , Johnson Howard M.
Published: April 2014

IFN-γ contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1α via a complex of IFN-γ/IFN-γ receptor (IFNGR)-1/STAT1α with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1α. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-γ resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-γ and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1α, but did not affect the binding of IFN-γ to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1α were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-γ, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-γ binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1α or the nuclear translocation of IFN-γ or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-γ-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-γ, IFNGR-1, and STAT1α.