bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression.
Authors: Bikoff E K , Huang L Y , Episkopou V , van Meerwijk J , Germain R N , Robertson E J
Published: June 2004

We used gene targeting techniques to produce mice lacking the invariant chain associated with major histocompatibility complex (MHC) class II molecules. Cells from these mice show a dramatic reduction in surface class II, resulting from both defective association of class II alpha and beta chains and markedly decreased post-Golgi transport. The few class II alpha/beta heterodimers reaching the cell surface behave as if empty or occupied by an easily displaced peptide, and display a distinct structure. Mutant spleen cells are defective in their ability to present intact protein antigens, but stimulate enhanced responses in the presence of peptides. These mutant mice have greatly reduced numbers of thymic and peripheral CD4+ T cells. Overall, this striking phenotype establishes that the invariant chain plays a critical role in regulating MHC class II expression and function in the intact animal.
Mycobacterial virulence. Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity.
Authors: North R J , Izzo A A
Published: June 2004

The kinetics of growth of two virulent strains of mycobacteria (M. tuberculosis Erdman and M. tuberculosis H37Rv) and two attenuated strains (M. tuberculosis H37Ra and M. bovis Bacillus Calmette-Guerin [BCG]) were studied in the lungs, livers, spleens, and kidneys of severe combined immunodeficient (SCID) mice and of their coisogenic CB-17 immunocompetent counterparts. It was found, in keeping with the findings of earlier investigators (Pierce, C. H., R. J. Dubos, and W. B. Schaefer. 1953. J. Exp. Med. 97:189.), that in immunocompetent mice, virulent organisms grew progressively only in the lungs, whereas the growth of attenuated organisms was controlled in all organs. In SCID mice, in contrast, virulent mycobacteria grew rapidly and progressively in all organs, as did BCG, although at a slower rate. However, H37Ra failed to grow progressively in any organs of SCID mice, unless the mice were treated with hydrocortisone. In fact, hydrocortisone treatment enabled virulent, as well as attenuated, organisms to grow strikingly more rapidly in all organs of SCID mice and in all organs of CB-17 mice. A histological study showed that in SCID mice, multiplication of mycobacteria in the liver occurs in the cytoplasm of macrophages in granulomas and presumably in macrophages in other organs. It is suggested, therefore, that the macrophages of SCID mice possess a glucocorticoid-sensitive mycobacterial mechanism that prevents virulent and avirulent mycobacteria from expressing their true minimal doubling times. In the absence of this mechanism in the lungs of hydrocortisone-treated SCID mice, the doubling times of Erdman, H37Rv, BCG, and H37Ra were 17.7, 17.4, 44.6, and 98.6 h, respectively. The possible importance of a rapid multiplication rate for mycobacterial virulence is discussed.
Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.
Authors: Xie Q W , Whisnant R , Nathan C
Published: June 2004

Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.
Tumor necrosis factor alpha cooperates with interleukin 3 in the recruitment of a primitive subset of human CD34+ progenitors.
Authors: Caux C , Durand I , Moreau I , Duvert V , Saeland S , Banchereau J
Published: June 2004

We have recently demonstrated that tumor necrosis factor alpha (TNF-alpha) potentiates interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor-induced growth of CD34+ hematopoietic progenitor cells (HPC), and favors the generation of dendritic/Langerhans cells. The stimulatory effect of TNF-alpha was detailed in the present study. Thus, CD34+ HPC entering in cycle (S/G2M) after a 48-h pulse with IL-3 expressed the transferrin receptor (TfR), and fluorescence-activated cell sorter-separated TfR+ HPC, but not TfR-HPC, showed a high proliferative response to IL-3. In contrast, TfR-HPC were found to undergo strong proliferation in response to IL-3 + TNF-alpha. Limiting dilution experiments indicated that TNF-alpha increased both the frequency and the average size of clones generated from TfR-HPC as a result of the development of a higher number of large clones. In contrast, TNF-alpha did not enhance the IL-3-dependent proliferation of TfR+ HPC. Preculturing CD34+ HPC for 48 h with TNF-alpha enhanced the subsequent generation of IL-3-dependent colony-forming units. Precultures with TNF-alpha or cultures with suboptimal doses of TNF-alpha allowed the recruitment of cells with both granulocytic and monocytic differentiation potential. Taken together, our results indicate that TNF-alpha recruits a subpopulation of CD34+ HPC hyposensitive to IL-3, with high proliferative capacity and some features of multipotential progenitors, that are likely to be more primitive than those responding to IL-3 alone.
Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes.
Authors: Schall T J , Bacon K , Camp R D , Kaspari J W , Goeddel D V
Published: June 2004

Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration-dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.
Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice.
Authors: Shankar A H , Titus R G
Published: June 2004

Several studies indicate that the outcome of experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm) is determined by immunological events occurring shortly after infection. These events lead to outgrowth of either protective CD4+ T cells in the C57BL/6 mouse, which cures, or exacerbative cells in the BALB/c mouse, which succumbs to disease. Potential factors influencing the outgrowth of protective or exacerbative T cells include antigen-presenting cells (APC), cytokines, and parasite antigens. An in vitro system, in which one could precisely control the factors shaping early events in the T cell response to Lm, would be very useful. To this end, we have examined the in vitro response of naive lymphocytes to Lm promastigotes. The data presented here show that Lm-specific CD4+ T cell receptor alpha/beta + T cells can be generated in vitro from spleen and lymph node cell populations of naive mice. Furthermore, they can be obtained from the CD44low (unprimed) population of T lymphocytes, indicating that in vitro priming occurs. The ability to generate these T cells is dependent on the presence of live parasites and is not due to a parasite-derived nonspecific T cell mitogen. Restimulation, as assayed by proliferation, requires APC bearing syngeneic I-A. Optimal restimulation of the in vitro derived T cells is achieved only when live promastigotes are used. The T cells do not proliferate in response to a frozen-and-thawed lysate of promastigotes, yet they exhibit mild reactivity to lysates prepared from heat-shocked promastigotes. Furthermore, they do not recognize two predominant antigens on the promastigote surface, lipophosphoglycan and gp63. T cells derived in vitro with Lm show crossreactivity with live L. donovani, less crossreactivity with live L. mexicana, and no crossreactivity with live Bacillus-Calmette-Guerin or live Brugia malayi microfilariae. Finally, these early T cells, whether derived from healing C57BL/6 or nonhealing BALB/c mice, produce interleukin 2 (IL-2), IL-4, and interferon gamma.
Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains.
Authors: Pauza M E , Rehmann J A , LeBien T W
Published: June 2004

Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow cytometry and were clonal by Southern blotting. Surprisingly, two of the mu/lambda-expressing cell lines contained both kappa alleles in germline configuration, and synthesis/expression of conventional lambda L chains was directly proven by immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in one of them. Thus, human fetal bone marrow B lineage cells harbor the capacity to make functional lambda L chain gene rearrangements without rearranging or deleting either kappa allele. A third unusual cell line, designated 30.30, was observed to coexpress cell surface kappa and lambda L chains associated with mu H chains. The 30.30 cell line had a diploid karyotype, a single H chain rearrangement, both kappa alleles rearranged, and a single lambda rearrangement. Immunoprecipitation/SDS-PAGE confirmed that 30.30 cells synthesized and expressed kappa and lambda L chains. Multiparameter flow cytometry was used to demonstrate the existence of kappa+/lambda+ cells in fetal bone marrow and fetal spleen at frequencies of 2-3% of the total surface Ig+ B cell population. The flow cytometry data was confirmed by two-color immunofluorescence microscopy. The existence of normal human B cells expressing cell surface kappa and lambda refutes the widely accepted concept that expression of a single L chain isotype is immutable. The kappa+/lambda+ cells may represent transients undergoing L chain isotype switching.
Lysis of major histocompatibility complex class I-bearing cells in Borna disease virus-induced degenerative encephalopathy.
Authors: Planz O , Bilzer T , Sobbe M , Stitz L
Published: June 2004

CD8+ as well as CD4+ T cells and macrophages are of crucial importance for the pathogenesis of Borna disease in rats. This virus-induced immunopathological disease of the brain is characterized by neurological symptoms in the acute phase and chronic debility associated with severe loss of brain tissue in the late stage. We demonstrate here the cytotoxic activity of T lymphocytes in the brain of intracerebrally infected rats. T cells isolated from the brain of infected rats lyse major histocompatibility complex (MHC) class I-bearing target cells in the absence of MHC class II. Borna disease virus (BDV)-infected syngeneic skin cells and astrocytes, the latter one of the relevant target cells in vivo, were significantly lysed whereas infected allogeneic target cells were not. Most relevant to the in vivo situation, primary brain cell cultures propagated from the hippocampus of BDV-infected rats containing considerable numbers of neurons were lysed in vitro. Blocking experiments using antibodies directed against MHC class I antigen provided further evidence for the presence and activity of classical cytotoxic T lymphocytes. Antibodies against MHC class II antigen did not influence lysis of skin target cells but had an effect on lysis of astrocytes at late time points. Lymphocytes isolated from spleen, peripheral blood, or lymph nodes did not show cytotoxic activity. These results verify, on the cellular level, earlier findings that strongly suggest the involvement of CD8+ T cells in brain cell lesions, resulting in brain atrophy long after infection of rats with BDV. This is further evidenced by the presence of CD8+ T cells in direct proximity to neuronal cell lesions. Interestingly, the cytolytic capacity, demonstrated in vitro and strongly correlated to organ destruction, does not result in elimination of the virus but the virus persists in the central nervous system.
Leukocyte extravasation into the pancreatic tissue in transgenic mice expressing interleukin 10 in the islets of Langerhans.
Authors: Wogensen L , Huang X , Sarvetnick N
Published: June 2004

Transgenic expression of interleukin 10 (IL-10) in the islets of Langerhans leads to a pronounced pancreatic inflammation, without inflammation of the islets of Langerhans and without diabetes. A scattered infiltration of macrophages (M pi) precedes localized accumulations of CD4+ and CD8+ T lymphocytes, B lymphocytes, and M pi. This recruitment of inflammatory cells to the pancreas is somewhat surprising, since the biological activities of IL-10 in vitro indicate that IL-10 is a powerful immunosuppressive cytokine. Since endothelial cells play a major role in leukocyte extravasation, we examined if vascular changes and extralymphoid induction of peripheral and mucosal type vascular addressins contributed to IL-10-induced homing of mononuclear cells to the pancreas. The endothelium lining small vessels was highly activated in areas of inflammation, as the endothelial cells became cuboidal, and exhibited increased expression of major histocompatibility complex class II (Ia), intercellular adhesion molecule 1, and von Willebrand Factor. Furthermore, induction of vascular addressins simultaneously with accumulation of mononuclear cells around islets and vessels indicated that the endothelial cells take on the phenotype of differentiated endothelium specialized for leukocyte extravasation. In conclusion, pancreatic inflammation and vascular changes are prominent in IL-10 transgenic mice. We hypothesize that IL-10, in addition to its immuno-inhibitory properties, is a potent recruitment signal for leukocyte migration in vivo. These effects are relevant for in vivo therapeutic applications of IL-10.
Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses.
Authors: Miller G T , Hochman P S , Meier W , Tizard R , Bixler S A , Rosa M D , Wallner B P
Published: June 2004

Accessory cell surface molecules, such as T cell antigen CD2 and its ligand lymphocyte function-associated antigen 3 (LFA-3; CD58), are critical costimulatory pathways for optimal T cell activation in response to antigens. Interaction of CD2 with cell surface LFA-3 not only increases T cell/accessory cell adhesion, but also induces signal transduction events involved in the regulation of T cell responses. In this report, we show that specific interactions of LFA-3 with CD2 can result in T cell unresponsiveness to antigenic or mitogenic stimuli in vitro. By deletion of certain regions of the extracellular domain of LFA-3, we localized the CD2 binding site to the first domain of LFA-3. We then demonstrated that a soluble, purified first domain-LFA-3/IgG1 fusion protein (LFA3TIP) interacts with CD2 and binds to the same CD2 epitope as purified multimeric or cell surface-expressed LFA-3. LFA3TIP inhibits tetanus toxoid, hepatitis B surface antigen, anti-CD3 mAb, Con A, and phytohemagglutinin P-induced T cell proliferation, as well as xenogeneic and allogeneic mixed lymphocyte reactions (MLR). Unlike anti-LFA-3 or anti-CD2 monoclonal antibodies (mAbs) which inhibit T cell responses by blocking LFA-3/CD2 binding, LFA3TIP is capable of rendering T cells unresponsive to antigenic stimuli in situations where T cell activation is independent of CD2/LFA-3 interactions. Furthermore, LFA3TIP, but not blocking anti-CD2 mAbs, is capable of inducing T cell unresponsiveness to secondary stimulation in allogeneic MLR. This inhibition of T cell responses by LFA3TIP occurs through a different mechanism from that of mAbs to LFA-3 or CD2.
Differential effects of growth hormone and prolactin on murine T cell development and function.
Authors: Murphy W J , Durum S K , Longo D L
Published: June 2004

DW/J dwarf mice have a defect in their anterior pituitary and are deficient in growth hormone (GH) and prolactin (PRL). These mice have been demonstrated previously to have a deficiency in CD4/CD8 double-positive thymocytes, which could be corrected by treatment of these mice with recombinant human GH. Since PRL has been implicated in T cell function and human GH can interact with the PRL receptor, DW/J dwarf mice were treated with either ovine GH (ovGH) (20 micrograms/d) or ovine PRL (ovPRL) (20 micrograms/d). The ovine hormones can only bind their own specific receptors in the mouse. After several weeks of treatment, it was found that these two hormones produced markedly contrasting effects on T cells. Phenotypic analysis of the lymphoid organs was performed by flow cytometry and the functional capability of the peripheral T cells was assessed by immunizing the mice and determining the extent of antigen-specific proliferation of T cells obtained from the draining lymph nodes or by determining splenic mitogen responses. The results indicated that ovGH administration to dwarf mice resulted in significant increases in thymic cellularity yet had little effect on peripheral T cell responses. In contrast, the administration of ovPRL resulted in a further decrease in thymic cellularity when compared with untreated dwarf mice. No thymic effects of either ovGH or ovPRL administration were detected on the normal +/? counterparts. However, ovPRL administration resulted in a significant increase in the number and function of antigen-specific peripheral T cells in both immunized dwarf and +/? mice. The adjuvant effects of PRL occurred even though the mice also received complete Freund's adjuvant. These results suggest that neuroendocrine hormones may act in concert in T cell development. GH appears to promote thymocyte proliferation, while PRL appears to decrease thymus size and yet augment the number and function of antigen-specific T cells in the periphery.
Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.
Authors: Chicz R M , Urban R G , Gorga J C , Vignali D A , Lane W S , Strominger J L
Published: June 2004

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.
Freezing adhesion molecules in a state of high-avidity binding blocks eosinophil migration.
Authors: Kuijpers T W , Mul E P , Blom M , Kovach N L , Gaeta F C , Tollefson V , Elices M J , Harlan J M
Published: June 2004

Leukocyte extravasation is mediated by multiple interactions of adhesive surface structures with ligands on endothelial cells and matrix components. The functional role of beta 1 (CD29) integrins (or very late antigen [VLA] proteins) in eosinophil migration across polycarbonate filters was examined under several in vitro conditions. Eosinophil migration induced by the chemoattractant C5a or platelet-activating factor was fully inhibited by monoclonal antibody (mAb) 8A2, a recently characterized "activating" CD29 mAb. However, inhibition by mAb 8A2 was observed only under filter conditions that best reflected the in vivo situation, i.e., when the eosinophils migrated over filters preincubated with the extracellular matrix (ECM) protein fibronectin (FN), or when the filters were covered with confluent monolayers of cultured human umbilical vein endothelial cells (HUVEC). When bare untreated filters were used, mAb 8A2 had no effect, whereas the C5a-directed movement was prevented by CD18 mAb. Studies with alpha-subunit (CD49)-specific mAbs indicated that the integrins VLA-4 and -5 mediated migration across FN-preincubated filters, and VLA-2, -4, -5, and -6 were involved in eosinophil migration through filters covered with HUVEC. In contrast with the activating CD29 mAb 8A2, a combination of blocking CD49 mAbs or the nonactivating but blocking CD29 mAb AIIB2 failed to inhibit completely eosinophil migration over FN-preincubated or HUVEC-covered filters. mAb 8A2 stimulated binding to FN but not to HUVEC. Moreover, eosinophil migration over FN-preincubated or HUVEC-covered filters was significantly inhibited by anti-connecting segment 1 (CS-1) mAbs, as well as the soluble CS-1 peptide (unlike migration across bare untreated filters). Thus, inhibition of eosinophil migration by mAb 8A2 depended upon the presence of ECM proteins and not upon the presence of HUVEC per se. In conclusion, "freezing" adhesion receptors of the beta 1 integrin family into their high-avidity binding state by the activating CD29 mAb 8A2 results in a complete inhibition of eosinophil migration under physiological conditions. Hence, activation of beta 1 integrin-mediated cell adhesion may represent a new approach to prevent influx of inflammatory cells.
Demonstration of large-scale migration of cortical thymocytes to peripheral lymphoid tissues in cyclosporin A-treated rats.
Authors: Zadeh H H , Goldschneider I
Published: June 2004

Young adult Lewis rats were maintained on diets containing 0.015 or 0.027% cyclosporin A (CSA) for periods of up to 6 wk. All animals showed complete depletion of medullary thymocytes (CD4+8- and CD4-8+, T cell receptor [TCR] alpha/beta hi, Thy-1med/low, terminal deoxynucleotidyl transferase negative [TdT-]) and a 50% reduction in the number of TdT- cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1med) within 1 wk of CSA treatment. In addition, about half of the animals displayed a 50% reduction in the number of TdT+ cortical thymocytes (CD4+8+, TCR alpha/beta low, Thy-1hi). These intrathymic changes were accompanied by a reciprocal increase in the number of double-positive (DP; CD4+8+) T cells in lymph nodes (LN) and spleens. To confirm that the latter T cells were recent thymic emigrants (RTE), CSA-treated rats were injected intrathymically with fluorescein isothiocyanate, and the phenotype of the labeled T cells appearing in LN was determined 16 h later. The results demonstrated that, in addition to those RTE exported in normal animals (> 90% medullary origin), the emigration of DP thymocytes, including large numbers of TdT+ thymocytes, was markedly increased. The presence of TdT+ cells, which normally do not leave the thymus, clearly identifies the DP RTE as originating from the thymus cortex. Intrathymic labeling studies also directly demonstrated that export of all thymocyte subsets ceases within 9 d of CSA treatment; and thymectomy experiments confirmed that the CSA-induced increase in phenotypically immature T cells resulted primarily from the disturbance of thymocyte maturation and emigration, rather than from a direct effect on preexisting T cells. These results suggest that a wave of cortical thymocytes, many of which presumably have not yet undergone negative selection, is released from the thymus during the first week of CSA treatment. The presence of these potentially unselected cells in peripheral lymphoid tissues may help to explain the increased frequency of autoreactive T cells observed in CSA-treated animals.
Antigen-driven B cell differentiation in vivo.
Authors: McHeyzer-Williams M G , McLean M J , Lalor P A , Nossal G J
Published: June 2004

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.
Intrathymic differentiation of V gamma 3 T cells.
Authors: Leclercq G , Plum J , Nandi D , De Smedt M , Allison J P
Published: June 2004

Whereas there is considerable information on the phenotypic and functional maturation of T cell receptor (TCR) alpha/beta thymocytes, comparatively little is known of the maturational processes that affect development of TCR-gamma/delta thymocytes. One class of gamma/delta T cells, those bearing the V gamma 3 gene product, are generated only during the early fetal stages of thymic development, and then migrate to the skin. Here we examine the intrathymic differentiation of these V gamma 3+ cells. The earliest V gamma 3 cells to appear in the thymus expressed low levels of TCR (V gamma 3low) and high levels of heat stable antigen (HSA). Over the next few days, V gamma 3+ thymocytes appeared which expressed high levels of TCR (V gamma 3high) and very low levels of HSA. The antigens CD5, CD45RB, and MEL14 were also differentially expressed on V gamma 3low versus V gamma 3high thymocytes, but the shift in expression was the opposite as compared with immature and mature TCR-alpha/beta thymocytes. Transfer experiments of sorted V gamma 3low/HSAhigh thymocytes to SCID thymic lobes showed that these cells were indeed the precursors of V gamma 3high/HSAlow thymocytes. The phenotype of the V gamma 3high thymocytes was similar to that of the postthymic V gamma 3+ cells found in the skin of adult mice. The differentiation of V gamma 3low in V gamma 3high thymocytes was also observed in fetal thymic organ culture. Addition of cyclosporin A (CsA) to these cultures had little effect on the appearance of V gamma 3low/HSAhigh cells, but blocked the appearance of V gamma 3high/HSAlow cells. These results show that, like alpha/beta T cells, V gamma 3+ thymocytes differentiate from TCRlow precursors to cells with a mature phenotype and that CsA inhibits this transition.
Molecular characterization of transgene-induced immunodeficiency in B-less mice using a novel quantitative limiting dilution polymerase chain reaction method.
Authors: Levinson D A , Campos-Torres J , Leder P
Published: June 2004

B-less mice express a human immunoglobulin (Ig) lambda transgene that induces a severe deficiency of both immature pre-B and mature B lymphocytes. To understand this perturbation in B lymphopoiesis, we have devised a sensitive limiting dilution polymerase chain reaction assay that quantitates specific Ig rearrangements and thus quantitates B lineage cells at various stages of differentiation within unfractionated bone marrow. We find that there are significantly reduced frequencies of both VH-to-DJH and VK-to-JK rearrangements in the transgenic strain, whereas the frequency of D-to-JH rearrangements approximates that of wild type. Since Ig gene rearrangements occur in a stepwise fashion in which D-to-JH joining precedes that of VH-to-DJH and VK-to-JK, these results indicate that the major block of B lymphocyte development in the B-less strain occurs after D-to-JH rearrangement. Interestingly, sequence analysis of residual VHDJH junctions from transgenic pre-B lymphocytes reveals that an abnormally high proportion of these are out of frame and therefore nonproductive. Taken together, these data suggest that early expression of the transgenic lambda protein specifically prevents the development of a normal-sized population of precursor B lymphocytes coexpressing functional IgH. The transgene-induced immunodeficiency appears to arise by a precocious maturation process in which precursors bypass a developmental stage associated with cellular expansion.
The structural basis of germline-encoded VH3 immunoglobulin binding to staphylococcal protein A.
Authors: Hillson J L , Karr N S , Oppliger I R , Mannik M , Sasso E H
Published: June 2004

The ability of human VH3 immunoglobulins (Ig) to bind to staphylococcal protein A (SPA) via their Fab region is analogous to the binding of bacterial superantigens to T cell receptors. The present report establishes the structural basis for the interaction of SPA and VH3 Ig. We have studied a panel of 27 human monoclonal IgM that were derived from fetal B lymphocytes. As such, these IgM were expected to be encoded by unmutated germline genes. Binding to SPA in ELISA occurred with 15 of 15 VH3 IgM, but none of 12 IgM from the VH1, VH4, VH5, or VH6 families. The VH sequences of the 27 IgM were derived from 20 distinct VH elements, including 11 from the VH3 family. Use of D, JH, and CL genes was similar among VH3 and non-VH3 IgM. A comparison of the corresponding VH protein sequences, and those of previously studied IgM, identified a probable site for SPA binding that includes VH3 residues in framework region 3 (FR3), and perhaps FR1 and 3' complementary determining region 2. The results thus demonstrate that among human IgM, specificity for SPA is encoded by at least 11 different VH3 germline genes. Furthermore, like the T cell superantigens, SPA likely binds to residues in the VH framework region, outside the classical antigen-binding site of the hypervariable loops.
Chemically modified antigen preferentially elicits induction of Th1-like cytokine synthesis patterns in vivo.
Authors: Yang X , Gieni R S , Mosmann T R , HayGlass K T
Published: June 2004

Differential activation of CD4+ T cell subsets in vivo leads to the development of qualitatively different effector responses. We identify an approach that allows selective activation of strongly Th1-dominated immune responses to protein antigens. Whereas in vivo administration of ovalbumin (OVA) induces cytokine synthesis that is neither Th1 nor Th2 dominated, administration of glutaraldehyde polymerized, high relative molecular weight OVA (OA-POL) leads to 20-fold increase in the ratio of interferon gamma (IFN-gamma)/IL-4 and IFN-gamma/IL-10 synthesis observed after short-term, antigen-mediated restimulation directly ex vivo. In contrast, concurrent in vivo administration of anti-IFN-gamma mAb and OVA or OA-POL results in marked increases in IL-4 and IL-10, and decreased IFN-gamma production, reflecting a polarization of the response towards a Th2-like pattern of cytokine synthesis. These observations may be useful in clinical settings including hypersensitivity, autoimmune diseases, and vaccine development where the ability to actively select specific patterns of cytokine gene expression would be advantageous.
Lysis of ovarian cancer cells by human lymphocytes redirected with a chimeric gene composed of an antibody variable region and the Fc receptor gamma chain.
Authors: Hwu P , Shafer G E , Treisman J , Schindler D G , Gross G , Cowherd R , Rosenberg S A , Eshhar Z
Published: June 2004

To expand the spectrum of recognition of effector lymphocytes and to redirect them towards predefined targets, we have altered the specificity of human tumor-infiltrating lymphocytes (TIL) through stable modification with chimeric receptor genes consisting of single-chain antibody variable regions linked to the gamma subunit common to the immunoglobulin (Ig)G and IgE Fc receptors. Using either hapten or ovarian carcinoma-specific monoclonal antibodies, we constructed chimeric receptor genes and retrovirally introduced them into CD8+ TIL. Redirected TIL specifically lysed trinitrophenyl-labeled Daudi or a human ovarian carcinoma cell line (IGROV-1), and secreted granulocyte/macrophage colony-stimulating factor upon stimulation with the appropriate antigen. This strategy may allow new approaches towards the adoptive immunotherapy of cancer in humans.
Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.
Authors: Fritzler M J , Hamel J C , Ochs R L , Chan E K
Published: June 2004

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.
Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha.
Authors: Kasama T , Strieter R M , Standiford T J , Burdick M D , Kunkel S L
Published: June 2004

Neutrophil (polymorphonuclear leukocyte [PMN]) sequestration is one of the histologic hallmarks of an acute inflammatory response. During the natural evolution of an inflammatory response, PMNs are often replaced by mononuclear cells. This shift in the elicitation of specific leukocyte populations usually occurs as the inflammatory lesion enters either the repair/resolution stage or progresses to a chronic inflammation. To elucidate a potential mechanism for the temporal change from predominantly PMN recruitment to the presence of monocytes, we postulated that PMNs could be a rich source of monocyte chemotactic factors. In our studies, we have identified a dose-dependent induction of monocyte chemotactic activity by PMNs treated with lipopolysaccharide (LPS; 1-100 ng/ml). Interestingly, this monocyte chemotactic activity was significantly attenuated in the presence of neutralizing anti-human macrophage inflammatory protein 1 alpha (MIP-1 alpha) antibodies. Moreover, immunolocalization studies demonstrated the expression of MIP-1 alpha by stimulated PMNs. These findings showed that a significant amount of PMN-derived monocyte chemotactic activity was attributable to MIP-1 alpha. Subsequent characterization of MIP-1 alpha steady-state mRNA and antigen expression demonstrated both a dose- and time-dependent production by LPS-treated PMNs. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a potent PMN activator, failed to induce the expression of MIP-1 alpha over a wide range of concentrations. However, PMNs stimulated in the presence of both LPS and GM-CSF resulted in a synergistic expression pattern for MIP-1 alpha. PMNs stimulated in the presence of both GM-CSF and LPS demonstrated an enhanced and prolonged expression for both MIP-1 alpha mRNA and antigen, as compared with LPS alone. Messenger RNA stabilization analyses demonstrated that MIP-1 alpha mRNA isolated from PMNs stimulated in the presence of GM-CSF and LPS had a prolonged mRNA t1/2, as compared with LPS alone. These findings support the notion that PMNs are capable of producing MIP-1 alpha in the presence of LPS, and that GM-CSF can influence this production through prolongation of MIP-1 alpha mRNA t1/2. The production of PMN-derived MIP-1 alpha, in association with the expression of appropriate adhesion molecules at a site of inflammation, may be one of the central events that contributes to the temporal shift from predominantly PMNs to monocytes during the evolution of inflammation.
Major histocompatibility complex class II-restricted presentation of an internally synthesized antigen displays cell-type variability and segregates from the exogenous class II and endogenous class I presentation pathways.
Authors: Loss G E , Elias C G , Fields P E , Ribaudo R K , McKisic M , Sant A J
Published: June 2004

Although reported examples of endogenous antigen (Ag) presentation by major histocompatibility complex (MHC) class II molecules have increased, the mechanisms governing this process remain poorly defined. In this communication, we describe an experimental system designed to examine the mechanisms governing class II presentation of internal Ag. Our target peptide is processed from a transmembrane protein constitutively expressed by a variety of nucleated cells (MHC class I, H-2Ld), is naturally displayed by MHC class II molecules in vivo, and is recognized by a class II-restricted, CD4+ T cell hybridoma. Our results indicate that presentation of the Ld target Ag is independent of its plasma membrane expression, may not involve endosomal proteolysis, and thus may be distinct from the classically defined class II presentation pathway. In addition, the observations that Ld presentation does not require a functional TAP-1 complex, is not blocked by invariant chain, and cannot utilize cytoplasmic forms of H-2Ld, suggest that a classical class I pathway is not involved in this presentation event. Finally, our data suggest that different cofactors participate in MHC class II presentation of exogenous and endogenous Ag, and that disparate Ag presenting cells, such as B, T, and pancreatic islet cells, may differentially express these two class II pathways of Ag presentation.
C1 esterase inhibitor gene expression in rat Kupffer cells, peritoneal macrophages and blood monocytes: modulation by interferon gamma.
Authors: Armbrust T , Schwögler S , Zöhrens G , Ramadori G
Published: June 2004

Kupffer cells (KC) represent the main part of the tissue macrophages. Beside phagocytosis of particulate material, involvement of KC in immunological and inflammatory reactions has been supposed. As C1 esterase inhibitor (C1-INH) is a serine protease inhibitor involved in such processes, the aim of this work was to study C1-INH synthesis in KC and, by comparison, in peritoneal macrophages (PM) and blood monocytes (MC) of the rat. C1-INH synthesis was studied on the protein level by biosynthetic labeling, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and on the RNA level by Northern blotting of total RNA or by in situ hybridization. KC were found to express C1-INH gene spontaneously. C1-INH synthesis represents 1.3 +/- 0.2% of total protein synthesis at day 1 of culture and the absolute amount each cell synthesis remains constant during the whole time in culture. Transcripts of C1-INH were detected both in freshly isolated and in cultured KC. In contrast, spontaneous C1-INH gene expression was not detectable in freshly isolated PM, but only in cultured PM. In MC, C1-INH was not detectable at any time, whatever. Treatment of the cells with interferon gamma increased C1-INH synthesis in KC and in PM and caused an induction of C1-INH synthesis in MC. The results suggest that constitutive C1-INH synthesis is a functional marker for mature tissue macrophages.
Inhibition of c-fes expression by an antisense oligomer causes apoptosis of HL60 cells induced to granulocytic differentiation.
Authors: Manfredini R , Grande A , Tagliafico E , Barbieri D , Zucchini P , Citro G , Zupi G , Franceschi C , Torelli U , Ferrari S
Published: June 2004

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.
Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development.
Authors: Galy A , Verma S , Bárcena A , Spits H
Published: June 2004

Studies of the most immature T cell progenitors in the human thymus have been hampered by the lack of markers and assays that define these cells. In this report we used a novel human fetal thymic organ culture system to determine the potential of T cell precursors isolated from human postnatal thymus, to differentiate into CD3+ thymocytes, and to investigate early stages of human T cell development. It was found that thymocytes that lack the markers CD3, CD4, and CD8 (triple negative [TN]) can differentiate in an allogeneic organotypic thymic culture. The capacity of TN thymocytes to differentiate was exclusively confined to the CD34+ population. CD34- TN thymocytes failed to differentiate in this system. In contrast, cloned lines of CD3- thymocytes could only be established from CD34- TN thymocytes. Five subsets of CD3- thymocytes were found with the following phenotype: CD1-TN, CD1+TN, CD1+CD4+CD8-, CD1+CD4+CD8 alpha+ beta-, and CD1+CD4+CD8 alpha beta+. These subpopulations expressed decreasing levels of CD34. The CD1-CD3- population expressed the highest levels of CD34 supporting the notion that this population is the most immature T cell precursor in the thymus, whereas the CD1+CD4+CD8 alpha+ beta+ which did not express CD34 seems to be the most mature of these CD3- populations. This notion is supported by the observations that CD34+ cells isolated from fetal liver, which differentiated into T cells in a FTOC, developed into CD3+ cells via CD1- and CD4+CD8- intermediates. Based on these data, we present a model of early stages in human intrathymic development.