bluuebunny/crossref_metadata_2025 huggingface dataset:
Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.
Authors: Boczkowski D , Nair S K , Snyder D , Gilboa E
Published: June 2004

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.
Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level.
Authors: Sornasse T , Larenas P V , Davis K A , de Vries J E , Yssel H
Published: June 2004

The development of CD4+ T helper (Th) type 1 and 2 cells is essential for the eradication of pathogens, but can also be responsible for various pathological disorders. Therefore, modulation of Th cell differentiation may have clinical utility in the treatment of human disease. Here, we show that interleukin (IL) 12 and IL-4 directly induce human neonatal CD4- T cells, activated via CD3 and CD28, to differentiate into Th1 and Th2 subsets. In contrast, IL-13, which shares many biological activities with IL-4, failed to induce T cell differentiation, consistent with the observation that human T cells do not express IL-13 receptors. Both the IL-12-induced Th1 subset and the IL-4-induced Th2 subset produce large quantities of IL-10, confirming that human IL-10 is not a typical human Th2 cytokine. Interestingly, IL-4-driven Th2 cell differentiation was completely prevented by an IL-4 mutant protein (IL-4.Y124D), indicating that this molecule acts as a strong IL-4 receptor antagonist. Analysis of single T cells producing interferon gamma or IL-4 revealed that induction of Th1 cell differentiation occurred rapidly and required only 4 d of priming of the neonatal CD4+ T cells in the presence of IL-12. The IL-12-induced Th1 cell phenotype was stable and was not significantly affected when repeatedly stimulated in the presence of recombinant IL-4. In contrast, the differentiation of Th2 cells occurred slowly and required not only 6 d of priming, but also additional restimulation of the primed CD4+ T cells in the presence of IL-4. Moreover, IL-4-induced Th2 cell phenotypes were not stable and could rapidly be reverted into a population predominantly containing Th0 and Th1 cells, after a single restimulation in the presence of IL-12. The observed differences in stability of IL-12- and IL-4-induced human Th1 and Th2 subsets, respectively, may have implications for cytokine-based therapies of chronic disease.
Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.
Authors: Alexander-Miller M A , Leggatt G R , Sarin A , Berzofsky J A
Published: June 2004

Experimental data suggest that negative selection of thymocytes can occur as a result of supraoptimal antigenic stimulation. It is unknown, however, whether such mechanisms are at work in mature CD8+ T lymphocytes. Here, we show that CD8+ effector cytotoxic T lymphocytes (CTL) are susceptible to proliferative inhibition by high dose peptide antigen, leading to apoptotic death mediated by TNF-alpha release. Such inhibition is not reflected in the cytolytic potential of the CTL, since concentrations of antigen that are inhibitory for proliferation promote efficient lysis of target cells. Thus, although CTL have committed to the apoptotic pathway, the kinetics of this process are such that CTL function can occur before death of the CTL. The concentration of antigen required for inhibition is a function of the CTL avidity, in that concentrations of antigen capable of completely inhibiting high avidity CTL maximally stimulate low avidity CTL. Importantly, the inhibition can be detected in both activated and resting CTL. Blocking studies demonstrate that the CD8 molecule contributes significantly to the inhibitory signal as the addition of anti-CD8 antibody restores the proliferative response. Thus, our data support the model that mature CD8+ CTL can accommodate an activation signal of restricted intensity, which, if surpassed, results in deletion of that cell.
Immune deviation of 2C transgenic intraepithelial lymphocytes in antigen-bearing hosts.
Authors: Guehler S R , Bluestone J A , Barrett T A
Published: June 2004

The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.
The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer.
Authors: Pende D , Biassoni R , Cantoni C , Verdiani S , Falco M , di Donato C , Accame L , Bottino C , Moretta A , Moretta L
Published: June 2004

Human natural killer (NK) cells express inhibitory receptors that are specific for different groups of HLA-C or HLA-B alleles. The majority of these receptors belong to the immunoglobulin (Ig) superfamily and are characterized by two or three extracellular Ig-like domains. Here we describe a novel inhibitory NK receptor that is specific for a group of HLA-A alleles. The HLA-A3-specific NK cell clone DP7 has been used for mice immunization. Two mAbs, termed Q66 and Q241, bound to the immunizing clone and stained only a subset of NK cell populations or clones. Among Q66 mAb-reactive clones, we further selected those that did not express any of the previously identified HLA-class I-specific NK receptors. These clones did not lyse HLA-A3+ (or -A11+) target cells, but lysis of these targets could be detected in the presence of Q66 or Q241 mAbs. On the other hand, target cells expressing other HLA-A alleles, including -A1, -A2, and -A24, were efficiently lysed. Moreover, none of the HLA-C or HLA-B alleles that were tested exerted a protective effect. Q66+, but not Q66- NK cell clones, expressed messenger RNA coding for a novel 3 Ig domain protein homologous to the HLA-C (p58) and HLA-B (p70) receptors. The corresponding cDNA (cl.1.1) was used to generate transient and stable transfectants in COS7 and NIH3T3 cell lines, respectively. Both types of transfectants were specifically stained by Q66 and Q241 mAbs. Since the cytoplasmic tail of Q66-reactive molecules was at least 11 amino acid longer than the other known p58/p70 molecules, we could generate an antiserum specific for the COOH-terminus of Q66-reactive molecules, termed PGP-3. PGP-3 immunoprecipitated, only from Q66+ NK cells, molecules displaying a molecular mass of 140 kD, under nonreducing conditions, which resolved, under reducing conditions, in a 70-kD band. Thus, differently from the other p58/p70 receptors, Q66-reactive molecules appear to be expressed as disulphide-linked dimers and were thus termed p140. The comparative analysis of the amino acid sequences of p58, p70, and p140 molecules revealed the existence of two cysteins proximal to the transmembrane region, only in the amino acid sequence of p140 molecules.
Regulation of pre-T cell receptor (pT alpha-TCR beta) gene expression during human thymic development.
Authors: Ramiro A R , Trigueros C , Márquez C , San Millán J L , Toribio M L
Published: June 2004

In murine T cell development, early thymocytes that productively rearrange the T cell receptor (TCR) beta locus are selected to continue maturation, before TCR alpha expression, by means of a pre-TCR alpha- (pT alpha-) TCR beta heterodimer (pre-TCR). The aim of this study was to identify equivalent stages in human thymocyte development. We show here that variable-diversity-joining region TCR beta rearrangement and the expression of full-length TCR beta transcripts have been initiated in some immature thymocytes at the TCR alpha/beta- CD4+CD8- stage, and become common in a downstream subset of TCR alpha/beta- CD4+CD8+ thymocytes that is highly enriched in large cycling cells. TCR beta chain expression was hardly detected in TCR alpha/beta- CD4+CD8- thymocytes, whereas cytoplasmic TCR beta chain was found in virtually all TCR alpha/beta- CD4+CD8+ blasts. In addition, a TCR beta complex distinct from the mature TCR alpha/beta heterodimer was immunoprecipitated only from the latter subset. cDNA derived from TCR alpha/beta- CD4+CD8+ blasts allowed us to identify and clone the gene encoding the human pT alpha chain, and to examine its expression at different stages of thymocyte development. Our results show that high pT alpha transcription occurs only in CD4+CD8- and CD4+CD8+ TCR alpha/beta- thymocytes, whereas it is weaker in earlier and later stages of development. Based on these results, we propose that the transition from TCR alpha/beta- CD4+CD8- to TCR alpha/beta- CD4+CD8+ thymocytes represents a critical developmental stage at which the successful expression of TCR beta promotes the clonal expansion and further maturation of human thymocytes, independent of TCR alpha.
Differing roles for B7 and intercellular adhesion molecule-1 in negative selection of thymocytes.
Authors: Kishimoto H , Cai Z , Brunmark A , Jackson M R , Peterson P A , Sprent J
Published: June 2004

To ensure self tolerance, immature thymocytes with high binding affinity for self peptides linked to major histocompatibility complex (MHC) molecules are eliminated in situ via apoptosis (negative selection). The roles of two costimulatory molecules, B7-1 and intercellular adhesion molecule-1 (ICAM-1), in negative selection was examined by studying apoptosis of T cell receptor transgenic CD4+8+ thymocytes cultured with specific peptides presented by MHC class I-transfected Drosophila cells. When coexpressed on these cells, B7-1 and ICAM-1 act synergistically and cause strong class 1-restricted negative selection of thymocytes. When expressed separately, however, B7-1 and ICAM-1 display opposite functions: negative selection is augmented by B7-1, but is inhibited by ICAM-1. It is notable that B7-1 is expressed selectively in the thymic medulla, whereas ICAM-1 is expressed throughout the thymus. Because of this distribution, the differing functions of B7-1 and ICAM-1 may dictate the sites of positive and negative selection. Thus, in the cortex, the presence of ICAM-1, but not B7-1, on the cortical epithelium may preclude or reduce negative selection and thereby promote positive selection. Conversely, the combined expression of B7-1 and ICAM-1 may define the medulla as the principal site of negative selection.
Retro-retinoids in regulated cell growth and death.
Authors: O'Connell M J , Chua R , Hoyos B , Buck J , Chen Y , Derguini F , Hämmerling U
Published: June 2004

Vitamin A serves as a prohormone from which three classes of active metabolites are derived: the aldehydes, the carboxylic acids, and the retro-retinoids. Although these three classes are united under the rubric of signal transduction, they act by different molecular mechanisms: the 11-cis-retinaldehydes combine with opsin to form the universal visual pigments and the retinoic acids form ligands for transcription factors, whereas the retro-retinoids, as shown here, intersect with signal transduction at a cytoplasmic or membrane site. The retro-retinoid, anhydroretinol (AR), has long been known to act as a growth inhibitor in lymphocytes, whereas 14-hydroxy-4,14-retro-retinol (14-HRR) is required for normal lymphocyte proliferation. A mutually reversible relationship exists between these two retro-retinoids as one can reverse the effects of the other when given in pharmacological doses. The common explanation for reversible inhibition is competition for a shared receptor. We now provide evidence that when AR is given to T cells unmitigated by 14-HRR, rapid cell death can occur. The circumstances are closely related to nonclassical forms of apoptosis: within 2 h of AR administration the T cells undergo widespread morphological changes, notably surface blebbing and ballooning and, inevitably, bursting. In contrast, nuclear changes are comparatively mild, as indicated by absence of chromatin condensation and overt DNA cleavage to discrete nucleosomal fragments, although DNA nicks are readily discernible by terminal deoxynucleotidyl transferase assay. What further distinguishes the AR-induced form of apoptosis from classical ones is a lack of requirements of messenger RNA and protein synthesis, suggesting that the events leading to cell death are primarily initiated and play themselves out in the cytoplasm. This view is further reinforced by the finding that herbimycin A can prevent the onset of programmed cell death. The importance of our findings is that they strongly suggest a second messenger role for vitamin A metabolites in the cytoplasmic realm that has not been seen previously. These findings are entirely compatible with a general notion that in a cell requiring multiple coordinated signals for survival, the provision of an unbalanced signal can initiate programmed cell death. Collectively, our data also challenge the paradigm that retinoids (outside vision) solely mediate their function via the steroid/ retinoic acid receptor family of nuclear transcription factors. Instead, a mode of action in the cytoplasmic realm akin to one attributed to other small lipophilic second messenger molecules, such as diacyl glycerol or ceramide, may apply to retro-retinoids.
Interleukin-2 regulates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes.
Authors: Loetscher P , Seitz M , Baggiolini M , Moser B
Published: June 2004

Several studies have shown that CC chemokines attract T lymphocytes, and that CD45RO+, memory phenotype cells are considered to be the main responders. The results, however, have often been contradictory and the role of lymphocyte activation and proliferation has remained unclear. Using CD45RO+ blood lymphocytes cultured under different stimulatory conditions, we have now studied chemotaxis as well as chemokine receptor expression. Expression of the RANTES/MIP-1 alpha receptor (CC-CKR1) and the MCP-1 receptor (CC-CKR2) was highly correlated with migration toward RANTES, MCP-1, and other CC chemokines, and was strictly dependent on the presence of IL-2 in the culture medium. Migration and receptor expression were rapidly downregulated when IL-2 was withdrawn, but were fully restored when IL-2 was added again. The effect of IL-2 could be partially mimicked by IL-4, IL-10, or IL-12, but not by IL-13, IFN gamma, IL-1 beta, TNF-alpha, or by exposure to anti-CD3, anti-CD28 or phytohemagglutinin. Activation of fully responsive lymphocytes through the TCR/CD3 complex and CD28 antigen actually had the opposite effect. It rapidly downregulated receptor expression and consequent migration even in the presence of IL-2. In contrast to the effects on CC chemokine receptors, stimulation of CD45RO+ T lymphocytes with IL-2 neither induced the expression of the CXC chemokine receptors, IL8-R1 and IL8-R2, nor chemotaxis to IL-8. The prominent role of IL-2 in CC chemokine responsiveness of lymphocytes suggests that IL-2-mediated expansion is a prerequisite for the recruitment of antigen-activated T cells into sites of immune and inflammatory reactions.
Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.
Authors: Zheng L M , Ojcius D M , Garaud F , Roth C , Maxwell E , Li Z , Rong H , Chen J , Wang X Y , Catino J J , King I
Published: June 2004

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell-depleted mice). However, IL-10 downregulation of pro-inflammatory cytokine production and/or recruitment of additional effector cells may also be involved in the anti-tumor effect at higher local concentrations of IL-10, since transfected B16 tumor cells expressing high amounts of IL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.
Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.
Authors: Song Q , Burrows S R , Smith G , Lees-Miller S P , Kumar S , Chan D W , Trapani J A , Alnemri E , Litwack G , Lu H , Moss D J , Jackson S , Lavin M F
Published: June 2004

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.
Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages.
Authors: Allen L A , Aderem A
Published: June 2004

It has long been known from the results of ultrastructural studies that complement- and immunoglobulin G (IgG)-opsonized particles are phagocytosed differently by macrophages (Kaplan. G. 1977. Scand. J. Immunol. 6:797-807). Complement-opsonized particles sink into the cell, whereas IgG-coated particles are engulfed by lamellipodia, which project from the cell surface. The molecular basis for these differences is unknown. We used indirect immunofluorescence and confocal microscopy to examine how cytoskeletal proteins associate with phagosomes containing complement-opsonized zymosan (COZ) particles or IgG beads in phorbol-myristateacetate-treated peritoneal macrophages. During ingestion of COZ, punctate structures rich in F-actin, vinculin, alpha-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface. These foci are detected beneath bound COZ within 30 s of warming the cells to 37 degrees C, and their formation requires active protein kinase C. By contrast, during Fc receptor-mediated phagocytosis, all proteins examined were uniformly distributed on or near the phagosome surface. Moreover, ingestion of IgG beads was blocked by tyrosine kinase inhibitors, whereas phagocytosis of COZ was not. Thus, the signals required for particle ingestion, and the arrangement of cytoskeletal proteins on the phagosome surface, vary depending upon which phagocytic receptor is engaged. Moreover, complement receptor (CR)-mediated internalization required intact microtubules and was accompanied by the accumulation of vesicles beneath the forming phagosome, suggesting that membrane trafficking plays a key role in CR-mediated phagocytosis.
Selectins and neutrophil traffic: margination and Streptococcus pneumoniae-induced emigration in murine lungs.
Authors: Mizgerd J P , Meek B B , Kutkoski G J , Bullard D C , Beaudet A L , Doerschuk C M
Published: June 2004

The roles of selectins in the pulmonary margination and emigration of neutrophils were investigated by using mice genetically deficient in both E- and P-selectins (E/P mutants) and/or by intravenous injections of fucoidin (inhibiting both L- and P-selectins). E/P mutants were neutrophilic (14.7 +/- 4.9 x 10(6) vs. 0.8 +/- 0.1 x 10(6) neutrophils/ml). This neutrophilia was associated with increased margination of neutrophils within pulmonary capillaries (39.7 +/- 9.4 vs. 4.6 +/- 1.1 neutrophil profiles per 100 red blood cell profiles) but no change in margination within noncapillary pulmonary microvessels. After intratracheal instillation of Streptococcus pneumoniae, lungs of E/P mutants displayed increased neutrophil emigration (564 +/- 92 vs. 116 +/- 19 neutrophils per 100 alveolar profiles), edema (5.3 +/- 1.5 vs. 1.5 +/- 0.4 microliter/g body weight), and histologic evidence of lung injury compared with those in wild-type (WT). Fucoidin treatment did not affect neutrophil emigration during streptococcal pneumonia in WT or E/P mice. During pneumonia, the number of white blood cells (WBC) tethered to or spread upon the noncapillary vessel endothelium increased in both WT and E/P lungs. These are the first data demonstrating that neutrophil margination in uninfected pulmonary capillaries does not require E- and P-selectins; that streptococcal pneumonia induces an E- and P-selectin-independent increase in WBC interactions with noncapillary endothelium; and that migration of neutrophils to alveoli can occur despite deficiency or inhibition of all of the known selectins.
Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).
Authors: Loftus D J , Castelli C , Clay T M , Squarcina P , Marincola F M , Nishimura M I , Parmiani G , Appella E , Rivoltini L
Published: June 2004

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).
The characteristic site-specific reactivation phenotypes of HSV-1 and HSV-2 depend upon the latency-associated transcript region.
Authors: Yoshikawa T , Hill J M , Stanberry L R , Bourne N , Kurawadwala J F , Krause P R
Published: June 2004

After replication at sites of initial inoculation, herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) establish lifelong latent infections of the sensory and autonomic neurons of the ganglia serving those sites. Periodically, the virus reactivates from these neurons, and travels centripetally along the neuronal axon to cause recurrent epithelial infection. The major clinically observed difference between infections with herpes simplex virus type 1 and type 2 is the anatomic site specificity of recurrence. HSV-1 reactivates most efficiently and frequently from trigeminal ganglia, causing recurrent ocular and oral-facial lesions, while HSV-2 reactivates primarily from sacral ganglia causing recurrent genital lesions. An intertypic recombinant virus was constructed and evaluated in animal models of recurrent ocular and genital herpes. Substitution of a 2.8-kbp region from the HSV-1 latency-associated transcript (LAT) for native HSV-2 sequences caused HSV-2 to reactivate with an HSV-1 phenotype in both animal models. The HSV-2 phenotype was restored by replacing the mutated sequences with wild-type HSV-2 LAT-region sequences. These sequences or their products must act specifically in the cellular environments of trigeminal and sacral neurons to promote the reactivation patterns characteristic of each virus.
CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development.
Authors: Gravestein L A , van Ewijk W , Ossendorp F , Borst J
Published: June 2004

CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti-CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3-mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.
Role of appendix in the development of inflammatory bowel disease in TCR-alpha mutant mice.
Authors: Mizoguchi A , Mizoguchi E , Chiba C , Bhan A K
Published: June 2004

T cell receptor-alpha mutant mice (TCR-alpha-/-), created by gene targeting of the TCR-alpha gene in embryonic stem cells, spontaneously develop inflammatory bowel disease (IBD) resembling human ulcerative colitis. Since gut-associated lymphoid tissue is likely to play an important role in the development of chronic intestinal inflammation, we examined the changes in the appendix lymphoid follicle (ALF) and Peyer's patches (PP) in these mice. We found the structure of the ALF to be remarkably similar to that of the PP in the small intestine; in both instances, lymphoid follicles covered by surface epithelium (dome-formation) were found. The amount of proliferation in the lymphoid follicles of the appendix estimated by in vivo incorporation of 5-bromo-2'deoxyuridine was more than two times that of PP in TCR-alpha-/- mice. ELISPOT assay showed an increase of IgA, IgG1, and IgG2a, but not IgM-secreting B cells in ALF of TCR-alpha-/- mice compared to TCR-alpha+/- control mice. Furthermore, TCR-alpha-/- mice revealed an increase of autoantibody-producing B cells against the cytoskeletal protein tropomyosin in ALF as compared to PP. When TCR-alpha-/- mice underwent appendectomy at a young age (3-5 wk), the number of mesenteric lymph nodes cells at 6-7 mo were markedly less than in the sham-operated TCR-alpha-/- mice. Furthermore, appendectomy at 1 mo of age suppressed the development of IBD, with only 3.3% of these mice developing IBD in the 6-7-mo period of observation. In contrast, approximately 80% of controls, including the sham-operated TCR-alpha-/- mice, developed IBD during this period. These results suggest that ALF, rather than PP, is the priming site of cells involved in the disease process and plays an important role in the development of IBD in TCR-alpha-/- mice.
The inter-locus recombinant HLA-B*4601 has high selectivity in peptide binding and functions characteristic of HLA-C.
Authors: Barber L D , Percival L , Valiante N M , Chen L , Lee C , Gumperz J E , Phillips J H , Lanier L L , Bigge J C , Parekh R B , Parham P
Published: June 2004

The vast majority of new human HLA class I alleles are formed by conversions between existing alleles of the same locus. A notable exception to this rule is HLA-B*4601 formed by replacement of residues 66-76 of the alpha 1 helix of B*1501 by the homologous segment of Cw*0102. This inter-locus recombination, which brings together characteristic elements of HLA-B and HLA-C structure, is shown here to influence function dramatically. Naturally processed peptides bound by B*4601 are distinct from those of its parental allotypes B*1501 and Cw*0102 and dominated by three high abundance peptides. Such increased peptide selectivity by B*4601 is unique among HLA-A,B,C allotypes. For other aspects of function, presence of the small segment of HLA-C-derived sequence in an otherwise HLA-B framework converts B*4601 to an HLA-C-like molecule. Alloreactive cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and cellular glycosidases all recognize B*4601 as though it were an HLA-C allotype. These unusual properties are those of an allotype which has frequencies as high as 20% in south east Asian populations and is associated with predisposition to autoimmune diseases and nasopharyngeal carcinoma.
High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.
Authors: Koch F , Stanzl U , Jennewein P , Janke K , Heufler C , Kämpgen E , Romani N , Schuler G
Published: June 2004

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.
Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation.
Authors: Cella M , Scheidegger D , Palmer-Lehmann K , Lane P , Lanzavecchia A , Alber G
Published: June 2004

We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels of bioactive IL-12. Other stimuli such as microbial agents, TNF-alpha or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregulating the expression of ICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-gamma production by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.
IL-12 unmasks latent autoimmune disease in resistant mice.
Authors: Segal B M , Shevach E M
Published: June 2004

Inbred mice exhibit a spectrum of susceptibility to induction of experimental allergic encephalomyelitis (EAE). We have compared the immune responses of the susceptible SJL (H-2s) and resistant B10.S (H-2s) strains to determine factors other than the MHC background which control resistance/susceptibility to EAE. The resistance of the B10.S strain was found to be secondary to an antigen-specific defect in the generation of Th 1 cells that produce IFN gamma. This defect in IFN gamma production could be restored by exposure of the myelin basic protein (MBP)-reactive T cells to IL-12 with the subsequent induction of the ability to transfer EAE to naive recipients. These findings have important implications for the therapeutic use of IL-12 and IL-12 antagonists and may explain the association between relapses/exacerbation of autoimmune disease and infectious diseases.
Early biochemical signals arise from low affinity TCR-ligand reactions at the cell-cell interface.
Authors: Beeson C , Rabinowitz J , Tate K , Gütgemann I , Chien Y H , Jones P P , Davis M M , McConnell H M
Published: June 2004

The kinetics of acid release by a mixture of T cells and antigen presenting cells were measured with a microphysiometer during a brief exposure to antigenic peptides. We find that some of the early biochemical events that lead to cellular proliferation cause a specific increase in the rate of acid release. The duration of this increase in acid release reflects the life-time of the peptide-MHC complexes. Peptides that form long-lived complexes produce a response that is stable for more than an hour. Serial TCR engagement is suggested by the observation that the amplitude of this stable response can be rapidly shifted up or down with additional agonist peptide or with antibodies that block T cell receptor binding. Cells briefly exposed to a peptide that forms short-lived peptide-MHC complexes produce a response that decays rapidly as peptide is washed away. A quantitative analysis of the kinetics of this decay in acidification demonstrates that intercellular TCR-ligand reactions are rapid, reversible, and of low apparent affinity with < 20% of peptide-MHC ligand bound to a TCR at any one time. These results demonstrate that the fraction of peptide-MHC ligands bound to TCRs at the cell-cell interface is no higher than anticipated from the affinities observed in solution for isolated TCRs and ligands.
CTLA-4: a negative regulator of autoimmune disease.
Authors: Karandikar N J , Vanderlugt C L , Walunas T L , Miller S D , Bluestone J A
Published: June 2004

CTLA-4, a CD28 homologue expressed on activated T cells, binds with high affinity to the CD28 ligands, B7-1 (CD80) and B7-2 (CD86). This study was designed to examine the role of CTLA-4 in regulating autoimmune disease. Murine relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) is a demyelinating disease mediated by PLP139-151-specific CD4+ T cells in SJL/J mice. Anti-CTLA-4 mAbs (or their F(ab) fragments) enhanced in vitro proliferation and pro-inflammatory cytokine production by PLP139-151-primed lymph node cells. Addition of either reagent to in vitro activation cultures potentiated the ability of T cells to adoptively transfer disease to naive recipients. In vivo administration of anti-CTLA-4 mAb to recipients of PLP139-151-specific T cells resulted in accelerated and exacerbated disease. Finally, anti-CTLA-4 treatment of mice during disease remission resulted in the exacerbation of relapses. Collectively, these results suggest that CTLA-4 mediates the downregulation of ongoing immune responses and plays a major role in regulating autoimmunity.
Fc gamma R-dependent mitogen-activated protein kinase activation in leukocytes: a common signal transduction event necessary for expression of TNF-alpha and early activation genes.
Authors: Trotta R , Kanakaraj P , Perussia B
Published: June 2004

Cross-linking the receptors for the Fc domain of IgG (Fc gamma R) on leukocytes induces activation of protein tyrosine kinases. The intermediary molecules that transduce to the nucleus the signals leading to induction of the diverse biological responses mediated by these receptors are not clearly identified. We have investigated whether mitogen-activated protein kinases (MAPK) are involved in transmembrane signaling via the three Fc gamma R present on monocytic, polymorphonuclear, and natural killer (NK) cells. Our results indicate that occupancy of Fc gamma RI and Fc gamma RII on the monocytic cell line THP-I and on polymorphonuclear leukocytes (PMN) induces, transiently and with fast kinetics, MAPK phosphorylation, as indicated by decreased electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and increased amounts of the proteins in antiphosphotyrosine antibody immunoprecipitates. This, associated with increased enzymatic activity, also occurs upon stimulation of the transmembrane isoform of CD16 (Fc gamma RIIIA) in NK cells and in a T cell line expressing transfected Fc gamma RIIIA alpha ligand-binding chain in association with zeta, but not upon stimulation of the glycosil-phosphatidylinositol-anchored Fc gamma RIIIB on PMN. Using the specific MAP kinase kinase inhibitor-PD 098059, we show that activation of MAPK is necessary for the Fc gamma R-dependent induction of c-fos and tumor necrosis factor alpha mRNA expression in monocytes and NK cells. These results underscore the role of MAPK as signal-transducing molecules controlling the expression of different genes relevant to leukocyte biology upon Fc gamma R stimulation.
Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells: implications for microbial attachment and oral vaccine targeting.
Authors: Frey A , Giannasca K T , Weltzin R , Giannasca P J , Reggio H , Lencer W I , Neutra M R
Published: June 2004

Transepithelial transport of antigens and pathogens across the epithelial barrier by M cells may be a prerequisite for induction of mucosal immunity in the intestine. Efficient transport of antigens and pathogens requires adherence to M cell apical surfaces. Coupling of antigen-containing particles to the pentameric binding subunit of cholera toxin (CTB) has been proposed as a means for increasing antigen uptake because the CTB receptor, ganglioside GM1, is a glycolipid present in apical membranes of all intestinal epithelial cells. To test the accessibility of enterocyte and M cell membrane glycolipids to ligands in the size ranges of viruses, bacteria, and particulate mucosal vaccines, we analyzed binding of CTB probes of different sizes to rabbit Peyer's patch epithelium. Soluble CTB-fluorescein isothiocyanate (diameter 6.4 nm) bound to apical membranes of all epithelial cells. CTB coupled to 14 nm colloidal gold (final diameter, 28.8 nm) failed to adhere to enterocytes but did adhere to M cells. CTB-coated, fluorescent microparticles (final diameter, 1.13 microns) failed to adhere to enterocytes or M cells in vivo or to well-differentiated Caco-2 intestinal epithelial cells in vitro. However, these particles bound specifically to GM1 on BALB/c 3T3 fibroblasts in vitro and to undifferentiated Caco-2 cells that lacked brush borders and glycocalyx. Measurements of glycocalyx thickness by electron microscopy suggested that a relatively thin (20 nm) glycocalyx was sufficient to prevent access of 1-micron microparticles to glycolipid receptors. Thus, the barrier function of the intestinal epithelial cell glycocalyx may be important in limiting microbial adherence to membrane glycolipids, and in CTB-mediated targeting of vaccines to M cells and the mucosal immune system.
Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice.
Authors: Mueller R , Krahl T , Sarvetnick N
Published: June 2004

Diabetes in nonobese diabetic (NOD) mice is a T cell-dependent autoimmune disease. The destructive activities of autoreactive T cells have been shown to be tightly regulated by effector molecules. In particular, T helper (Th) 1 cytokines have been linked to diabetes pathogenesis, whereas Th2 cytokines and the cells that release them have been postulated to be protective from disease. To test this hypothesis, we generated transgenic NOD mice that express interleukin (IL) 4 in their pancreatic beta cells under the control of the human insulin promoter. We found that transgenic NOD-IL-4 mice, both females and males, were completely protected from insulitis and diabetes. Induction of functional tolerance to islet antigens in these mice was indicated by their inability to reject syngeneic pancreatic islets and the failure of diabetogenic spleen cells to induce diabetes in transgenic NOD-IL-4 recipients. Interestingly, however, islet expression of IL-4 was incapable of preventing islet rejection in overtly diabetic NOD recipient mice. These results demonstrate that the Th2 cytokine IL-4 can prevent the development of autoimmunity and destructive autoreactivity in the NOD mouse. Its ability to regulate the disease process in the periphery also indicates that autoimmune diabetes in NOD mice is not a systemic disease, and it can be modulated from the islet compartment.
Involvement of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus.
Authors: Hattori N , Kawamoto H , Fujimoto S , Kuno K , Katsura Y
Published: June 2004

Flow cytometric and immunocytochemical analyses of murine fetal thymus (FT) cells with antibodies to various surface markers and transcription factors reveal that the synthesis of TCF-1 and GATA-3 protein begins simultaneously in a fraction of the most immature population of FT cells, which have the phenotype of CD4-CD8-CD44+CD25-. No TCF-1-producing cells is found in the fetal liver (FL). In CD44+CD25- FT cells, the production of TCF-1 is immediately followed by intracellular expression of CD3 epsilon. It is also found that the T cell development from FL, but not FT, progenitors in the FT organ culture system is severely inhibited by the addition of antisense oligonucleotides for either TCF-1 or GATA-3. These results strongly suggest that TCF-1 and GATA-3 play essential roles in the initiation of the earliest steps of T cell development in the thymus.
A tumor-suppressor function for Fas (CD95) revealed in T cell-deficient mice.
Authors: Peng S L , Robert M E , Hayday A C , Craft J
Published: June 2004

Fas (CD95) and its ligand are central regulatory molecules in hematopoietic cells. Previous studies have suggested a role for Fas in the regulation of tumor progression, but Fas has not yet been conclusively identified as a tumor suppressor. Fas-deficient individuals lack malignant tumors, perhaps because of regulation by T cells. To investigate such a possibility, mice deficient in both T cells and Fas were generated, and they were found to develop severe B cell dysregulation characterized by malignant, lethal B cell lymphoma. Lymphoma arose from a monoclonal B220+CD19-CD5-CD23- B cell secreting immunoglobulin M, kappa rheumatoid factor. In contrast, animals containing alpha beta T cells, gamma delta T cells, and/or functional Fas suppressed the development of lymphoma. These data indicate that Fas functions as a tumor suppressor, and identifies roles for both alpha beta T cells and gamma delta T cells in Fas-independent tumor regulation.