bluuebunny/crossref_metadata_2025 huggingface dataset:
Suicide selection of murine T helper clones specific for variable regions of the influenza hemagglutinin molecule
Authors: Brian Thomas D. , Skehel John J. , Mills Kingston H. G. , Graham Christine M.
Published: February 2007

A negative selection procedure has been developed to obtain murine T helper clones specific for variable regions of the influenza A hemagglutinin. T cell lines, established from mice primed by intranasal infection with X31 (H3N2) virus, were cross‐stimulated with natural variant viruses of known primary sequence (either A/TEXAS/1/77 or A/ENG878/69) and proliferating cells eliminated by treatment with the cell cycle‐ specific drug 5‐bromodeoxyuridine. After two suicide cycles, T cell lines were subtype specific and failed to recognize the natural variants. Clones were established by limiting dilution and their specificity was determined against a panel of viruses. Extensive diversity was evident in the reactivity of clones from individual donors, and two major T cell recognition sites were defined in the globular head region of the hemagglutinin molecule.
IgE receptors on human lymphocytes I. Identification of the molecules binding to monoclonal anti‐Fcε receptor antibodies
Authors: Nakajima Toshiharu , Delespesse Guy
Published: February 2007

The present study indicates that the surface molecules from Fcε receptor‐bearing human lymphoblastoid cells binding to monoclonal antibodies to Fcε receptor (mAbER) are identical to those binding to IgE. mAbER identify three components in the Nonidet‐P40 cell lysate of surface‐iodinated RPMI 8866 cells with approximately 65–95 kDa, 45 kDa and 37 kDa as estimated by sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis under reducing conditions and autoradiography. The same pattern is observed under nonreducing conditions, except for the low mol. wt component which displays an apparent molecular mass 31 kDa. When the labeled and solubilized membranes are adsorbed on IgE‐Sepharose the 45‐kDa component is always found in the eluate, whereas the 65–95‐kDa component is occasionally detected and the low mol. wt component is rarely found. The binding of these molecules to IgE‐Sepharose is inhibited by soluble IgE, mAbER but not by an unrelated mAb; reciprocally, the binding to mAbER‐Sepharose is prevented by mAbER and to some extent by IgE. To improve the stability of IgE‐FcεR complexes, biotinylated IgE was cross‐linked on surface iodinated intact cells, which were then solubilized and adsorbed on avidin‐Sepharose. Under these conditions, it was clearly shown that IgE binds to the same three surface molecules as mAbER. The isoelectric point of the high mol. wt component ranged from 4.2 to 4.4 as compared to 5.1–5.2 for the 45‐kDa and the 37‐kDa components. The observation in the Western blot assay that both the 65–95‐kDa and the 45‐kDa molecules react with 9 different mAbER indicates that these molecules have a polypeptide sequence in common.
IgE receptors on human lymphocytes II. Detection of cells bearing IgE receptors in unstimulated mononuclear cells by means of a monoclonal antibody
Authors: Delespesse Guy , Sarfati Marie , Rubio‐Trujillo Manuel , Wolowiec Terry
Published: February 2007

A monoclonal antibody (mAb) specific for lymphocyte IgE receptors (ER) was employed in a rosette assay for the detection of cells bearing IgE receptors (FcεR). The specificity of the assay was documented by inhibition studies with soluble immunoglobulins (Ig) and anti‐Ig antibodies. Moreover, similar results were obtained by employing the F(ab')2 fragment of mAbER instead of intact molecule. Circulating mononuclear cells isolated from normal or allergic adults and from umbilical cord blood contained approximately 8% of FcεR‐bearing cells with values ranging from 0.3 to 17%. Tonsillar lymphocytes contained about 30% of FcεR+ cells. After the removal of adherent cells, there was a small but significant reduction of the proportion of FcεR+ cells. When mononuclear cells were separated into T and B cell fractions by two‐cycle rosetting with 2‐aminoethylisothiouronium bromide hydrobromide‐treated sheep red blood cells, most of the FcεR+ cells were in the B cell fraction; however, a small proportion of FcεR+ was also found in the enriched T cells and double‐labeling experiments confirmed that these cells were indeed T lymphocytes. FcεR+ cells were purified by rosetting with mAbER‐coated erythrocytes and their phenotype was compared to that of FcεR− cells; FcεR+ cells contained about 90% of B cells (B1+) together with a small proportion of OKT3+, Leu 7+ and Mo2+cells. The bulk of T cells, macrophages and natural killer (NK) cells was found in the FcεR− cells which contained fewer B cells than the fraction of FcεR+ cells. These data thus indicated that the great majority of FcεR‐bearing cells are B cells but that a small proportion of NK cells, macrophages and T lymphocytes also express FcεR. Upon incubation at 37°C, B cells lost their FcεR and this phenomenon was selectively inhibited by IgE; however, purified T cells seemed to express more FcεR after overnight incubation at 37°C and this was not influenced by IgE. It is finally shown that the expression of FcεR is cyclic and that FcεR‐bearing B cells do not represent a functionally distinct subpopulation of B lymphocytes.
Monoclonal antibodies against idiotypic determinant(s) of the T cell receptor from HPB‐ALL cells induce IL 2 production in Jurkat cells without apparent evidence of binding
Authors: Carrel Stefan , Giuffrè Laura , Vacca Angelo , Salvi Suzanne , Mach Jean‐Pierre , Isler Patrick
Published: February 2007

Two monoclonal antibodies (mAb) directed against idiotypic determinants of the T cell receptor (anti‐Ti) from HPB‐ALL cells induce interleukin 2 (IL2) production in Jurkat T cells without evidence of binding to these cells as judged by fluorescence‐activated cell sorter (FACS) analysis, indirect antibody‐binding radioimmunoassay and direct binding studies with 125I‐labeled mAb. The IL2 response induced by these mAb observed both in the presence and absence of phorbol myristate acetate was in the range of that obtained when Jurkat cells were stimulated with phytohemagglutinin or anti‐T3 mAb (Leu 4).

The idiotypic specificity of the two anti‐HPB‐ALL Ti mAb was demonstrated by several criteria. Both mAb bound specifically to HPB‐ALL cells as determined by radioimmunoassay or FACS analysis but not with 8 other T cell lines. The anti‐HPB‐ALL Ti mAb precipitated a disulfide‐linked heterodimer of 85 kDa only from 125I‐labeled HPB‐ALL cells and not from other cell lines tested. Incubation of HPB‐ALL cells with anti‐T3 abrogated the expression of T3 and induced co‐modulation of the idiotypic structures detected by the two anti‐HPB‐ALL Ti mAb. Conversely, incubation of HPB‐ALL cells with either one of the anti‐Ti mAb abrogated the expression of T3 and of the idiotypic structures.

Our results suggest that mAb with an apparent unique specificity for the receptor of the immunizing T cell line HPB‐ALL can activate Jurkat cells by a very weak cross‐reaction with these cells, which is not detectable by conventional binding tests.
Antigen‐induced changes in B cell subsets in lymph nodes: analysis by dual fluorescence flow cytofluorometry
Authors: Kraal Georg , Hardy Richard R. , Gallatin William M. , Weissman Irving L. , Butcher Eugene C.
Published: February 2007

Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two‐color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and absolute number of IgM+, IgD+ B cells, which is followed by the formation of germinal centers. Germinal center cells, as soon as they appear on day 3 after primary immunization, bind high levels of peanut agglutinin, bear low levels of surface IgM but no detectable surface IgD, and are characterized by lack of staining with MEL‐14, a monoclonal antibody which recognizes a lymphocyte surface receptor involved in lymphocyte homing. The level of I‐A and H‐2K region‐encoded surface antigens on early germinal center cells is higher than on PNAlo B cells. During the first 7 days of the germinal centers there is a progressive decrease in the average level of H‐2K but not of Ia antigens. A similar decrease was observed for ThB. It is confirmed that the germinal center cell population contains the majority of antigen‐binding cells in the stimulated lymph node. These findings indicate that B cells are recruited nonspecifically to antigen‐stimulated lymph nodes, and that the antigen‐specific cells then selectively participate in the formation of germinal centers where they undergo specific differentiation events.
The T lymphocyte response to syngeneic λ2 light chain idiotopes. Significance of individual amino acids revealed by variant λ2 chains and idiotope‐mimicking chemically synthesized peptides
Authors: Hannestad Kristian , Kristoffersen Gunn , Briand Jean Paul
Published: February 2007

In the present study we have investigated the structure of the helper T cell (Th)‐defined idiotope (Id) of myeloma protein 315 λ2 light chain (λ2315) in BALB/c (H‐2d) mice which carry a high‐responder immune response gene for this Id. Three eptides were synthesized which spanned the third hypervariable region (HV3) of λ2315: peptides 88–99, 94–108 and 91–108. Only peptide 91–108 was capable of eliciting carrier‐specific Th that recognized M315 or free λ2315. These Th did not recognize λ25–7 chain which differs from λ2315 at 4 positions in this region; these are Tyr94, Ser95, Thr96, Tyr98 for λ25–7 and Phe94, Arg95, Asn96, Phe98 for λ2315. Immunization with peptide analogues revealed that substitution of Tyr for Phe94 was compatible with Id‐λ2315 mimicry, but substitution of Ser for Arg95 or Thr for Asn96 destroyed the Th‐recognized Id. Furthermore, Th primed with λ25–7 chain did not cross‐react with λ2T952 these λ2 chains only differ from each other at positions 98 and 99 at the Vλ2‐Jλ2 junction. The data indicate that individual amino acids of short peptide segments are critical for Th‐recognized Id of the λ2 HV3 loop and Vλ2‐Jλ2 junction. Furthermore, the immunogenicity of a small peptide suggests that the carrier (λ2)‐specific Th recognize Id that have been processed by antigen‐presenting cells (APC). This implies the existence of two categories of “internal images” of foreign or of self antigens: (a) serologically defined and (b) T lymphocyte defined. We propose that as a rule, Id processing by APC, including B cells, destroys the first and reveals the second category. The possible physiological function of these Id‐specific T cells in network interactions with B cells is discussed.
Immunity to lymphocytic choriomeningitis virus in B cell‐depleted mice: evidence for B cell and antibody‐independent protection by memory T cells
Authors: Cerny Andreas , Huegin Ambros W. , Sutter Stefan , Bazin Hervié , Hengartner Hans H. , Zinkernagel Rolf M.
Published: February 2007

Immunity against lymphocytic choriomeningitis virus (LCMV) in anti‐IgM‐treated B cell‐depleted mice was evaluated. We found that the following immune phenomena were independent of antibodies: (a) the generation of virus‐specific cytotoxic T cells; (b) the footpad swelling response against locally injected LCMV; (c) natural killer cell activity basic levels or after LCMV or poly(1). poly(C) stimulation; (d) immunopathologically mediated LCM after primary intracerebral inoculation; (e) immunological memory in LCMV‐immune mice assessed by immune protection against LCM after intracerebrally injected virus or as resistance against the local footpad swelling response to LCMV. This study demonstrates that humoral immunity plays no crucial role in immune protection and immunopathology in murine LCMV infection and suggests that protective memory T cell function is B cell and antibody independent.
Fine specificity and idiotype expression of antiphosphorylcholine IgE and IgG antibodies
Authors: Weber Ernst A. , Heusser Christoph H. , Blaser Kurt
Published: February 2007

The immune response to the phosphorylcholine (PC) hapten elicited in BALB/c mice by PC‐keyhole limpet hemocyanin (KLH) is composed of 2 groups of antibodies with specificity to PC and phenyl‐PC, respectively. They were designated as group I and group II anti‐PC antibodies. In this report we demonstrate that anti‐PC IgE antibodies elicited by PC‐KLH or PC‐ovalbumin belong to the group II and do not express the T15 idiotype. Anti‐PC IgG1, IgG2a, and IgG2b antibodies express group I characteristics in the primary response and bear the T15 idiotype. Later, after 5 weeks and 3 injections of PC‐KLH or PC‐ovalbumin, a change in these isotypes to group II antibodies is observed. In contrast, anti‐PC IgE is a group II antibody throughout progression of the immune response. The regulation of group I and group II antibody expression in serum is independent of the genetic background of the animals.
Multiple pathways for antigen‐independent activation of a T helper hybridoma
Authors: Torbett Bruce E. , Skidmore Barry , Clark William R.
Published: February 2007

An important question in mitogen activation of T cells is whether the T cell must interact with a major histocompatibility complex product during the activation process. The T helper hybridoma AODH 7.1 is specific for human gamma globulin in the context of IEd, and when activated secretes interleukin 2. The mitogen concanavalin A (Con A) can activate AODH 7.1 cells directly, but two other standard T cell mitogens, phytohemagglutinin (PHA) and neuraminidase‐galactose oxidase, cannot. However, Con A, PHA and neuraminidase‐galactose oxidase could all activate AODH 7.1 when presented on various cloned class II+ cell lines. There was an absolute requirement for the presentor cell to be class II+, and the activation signal on mitogen‐treated class II+ presentor could be blocked by monoclonal antibody to the class II antigens. To determine if class II molecules were absolutely required for the presentation of a mitogen activation signal we used class II− LtK− L cells and two LtK− cell class II gene transformants as presentor cells. Only the class II+ transformants, but not the class II− LtK− cells, could present either Con A or PHA. The class II requirement cannot be bypassed through provision of soluble factors. However, when Con A was used to activate AODH 7.1 cells directly, it appeared to be acting in a transmembrane fashion. It was not the case that AODH 7.1 cells were responding to Con A on a neighboring AODH 7.1 cell, and no class II antigens were involved. These results are consistent with the idea that different routes of activation exist for T cells, at least at the level of signal recognition.
Effect of anti‐Ia treatment on the production of anti‐DNA antibody by NZB mice
Authors: Klinman Dennis M. , Lefkowitz Michael D. , Raveche Elizabeth S. , Steinberg Alfred D.
Published: February 2007

A cell transfer system was used to study the effect of anti‐Ia antibodies on anti‐DNA‐producing B cells. B cells from autoimmune New Zealand Black (NZB) mice were necessary and sufficient to transfer anti‐DNA antibody production to congenic NZB. xid recipients. Anti‐Ia treatment of either donors or recipients led to a significant reduction in the number of B cells secreting anti‐DNA antibody. This effect was detectable after as little as 3 days of treatment and persisted for at least 1 month after the cessation of therapy. In this system, we could find no evidence of suppressor cell induction. These data suggest that anti‐Ia antibodies directly suppress autoantibody‐producing B lymphocytes.
The immune response to bacterial dextrans III. Ontogenic development and strain distribution of specific clonal precursors
Authors: Lundkvist Inger , Holmberg Dan , Ivars Fredrik , Coutinho Antonio
Published: February 2007

The frequencies of B512 dextran (Dex)‐specific B cell precursors were determined by limiting dilution analysis in a number of mouse strains originally described as “high responder”, “low responder” and “nonresponder” to this antigen. No significant difference in the frequencies of Dex‐specific precursors was found in C57BL/6, B10.BR, C3H/Tif, BALB/c and A/Sn adult mice. Together with the large intra‐strain variability in the magnitude of anti‐Dex PFC responses in vivo, these results established that differential reactivity in vivo cannot be ascribed to genetically controlled absence or wide variation in the frequency of Dex‐specific immunocompetent precursors.

A similar analysis of the Dex‐specific precursor frequency was carried out in C57BL/6 mice between 1 week and 3 months of age. While no Dex‐specific antibody response was detected in vivo before the age of 3 weeks, clonal precursor analysis revealed that the appearance of these specificities parallels the development of competent (IgM‐ producing) B lymphocyte clonal precursors, such that no significant difference in absolute frequencies of Dex‐specific precursors could be observed among these age groups. This is interpreted to suggest that the late development of the Dex‐specific antibody responses is regulatory rather than due to late rearrangement and activation of the appropriate V genes and a sequential expression of antibody specificities in ontogenic development.
Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule
Authors: Dalchau Rosemarie , Flanagan Brian F. , Fabre John W.
Published: February 2007

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage‐restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180‐kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage‐restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent‐solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase‐treated Daudi cells yielded a 130‐kDa lentil lectin‐binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDA) membrane proximal domain containing both of the common LC determinants and at least one N‐linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.
Inhibition of lipopolysaccharide‐induced monocyte interleukin 1 secretion by gangliosides
Authors: Cavaillon Jean‐Marc , Fitting Catherine
Published: February 2007

Gangliosides known to be potent immunosuppressors are shown to inhibit the secretion of interleukin 1 (IL1) by human monocytes stimulated with lipopolysaccharide (LPS). The inhibitory activity was observed either with mono‐, di‐ or trisialogan‐gliosides. The inhibition of LPS‐induced IL 1 secretion was obtained in the presence of indomethacin, indicating that the ganglioside inhibitory capacity was not due to prostaglandin induction. Indeed the inhibitory activity seems to be mediated by an interaction with LPS molecules, preventing their ability to deliver a signal to monocytes for IL 1 secretion.
IgE receptors on human lymphocytes III. Expression of IgE receptors on mitogen‐stimulated human mononuclear cells
Authors: Delespesse Guy , Sarfati Marie , Rubio‐Trujillo Manuel , Wolowiec Terry
Published: February 2007

Human peripheral blood mononuclear cells (PBMC) were tested for the expression of Fee‐receptor (FcεR) after stimulation with various mitogens in the absence of IgE. FcεR were found on virtually all the cells from 19 Epstein‐Barr virus‐transformed B cell lines including those derived from cord blood, from one agamma‐globulinemic patient and VDS‐0 pre‐B cells. Hence, the data clearly indicate that FcεR may be expressed on very immature B cells. PBMC cultures stimulated with either pokeweed mitogen (PWM), phytohemagglutinin (PHA) or concanavalin A displayed an early increase of their content in FcεR‐bearing cells followed by a decrease to levels below those of the control cultures. After fractionation of the PWM‐stimulated cultures into T and B cell‐enriched preparations, most of the FcεR+ cells were in the B cell fractions and the same low levels of FceR+ cells were found in the T cell fractions isolated from the PWM‐stimulated and from the control cultures. Double‐labeling experiments, employing biotinylated F(ab')2 monoclonal antibody to FcR and either fluorescein isothiocyanate‐conjugated B1 or Mo2 monoclonal antibodies, indicated that PWM mainly exerted its effect on B cells and on monocytes. This effect was T cell dependent and it was mediated by soluble factors of T cell origin. At the peak of the PHA or concanavalin A response, most of the FceR‐bearing cells were found in the B cell fraction but the T cells isolated from mitogen‐stimulated cultures contained significantly more FcεR+ cells than those from the control cultures, suggesting that T cell mitogens had increased the expression of FcεR on some T cells. This view was supported by the finding of a higher proportion of FceR+ cells in PHA‐stimulated than in control cultures of highly purified T cells with a maximum response at the end of the culture period. Double‐labeling experiments at the peak (day 2) of the peripheral blood mononuclear cell response indicated that the expression of FcεR was increased on B cells (Bl+) and on monocytes (Mo2+). By using the same approach at the peak of the T cell response (day 7), it was found that T cells isolated from PHA‐stimulated cultures expressed more FcεR than those isolated from control cultures. Moreover, in unstimulated cultures, FcεR was mainly expressed on T helper cells (Leu 3+) whereas in PHA‐stimulated cultures FcεR was expressed on both T helper and T suppressor/cytotoxic cells (Leu 2+).
Demonstration of C3b receptor‐like activity and of decay‐accelerating factor‐like activity on rabbit erythrocytes
Authors: Horstmann Rolf D. , Muller‐Eberhard Hans J.
Published: February 2007

C3b receptor activity and decay‐accelerating activity for the C3 convertase, C3b, Bb, were demonstrated on rabbit erythrocytes (erab). Particles bearing C3b agglutinated ERAB if the C3b was of rabbit origin, but not if it was of human origin. The reactivity of rabbit C3b was abolished by enzymatic conversion of C3b to C3bi. Deposited on ERAB. rabbit C3b, but not human C3b, was cleaved by factor I in the absence of factor H. Similarly, decay acceleration of the C3b, Bb complex on ERAB occurred when the complex was of rabbit origin, but not when it was of human origin. Soluble immune complexes in the presence of rabbit serum were bound to and then released from the erab. Binding of the complexes is presumed to be mediated by immune complex‐bound C3b and release to be the consequence of degradation of C3b by Factor I. These findings suggest that ERAB, like human erythrocytes, are endowed with membrane‐associated complement regulators that protect these cells against homologous complement attack and participate in the clearing mechanism of circulating immune complexes.
Azacytidine‐induced reactivation of adenosine deaminase in a murine cytotoxic T cell line
Authors: Castellazzi Marc , Vielh Philippe , Longacre Shirley
Published: February 2007

In C57BL/6 mice, cytotoxic T lymphocyte (CTL) lines have previously been found to exhibit low (< 150 U/mg) or undetectable (< 20 U/mg) adenosine deaminase (ADA) levels (Minkowski, Castellazzi and Buttin, J. Immunol. 1984. 133: 52) in contrast to what has been seen in T helper lines (1770 ± 340 U/mg; Minkowski and Bandeira, Cell. Immunol. 1985. 95: 380). Treatment of one of these CTL ADA− lines with the demethylating agent 5‐azacytidine gave rise to an ADA+ population. Subsequent cloning allowed the recovery of pure ADA+ clones showing a rather narrow range of activity with an average value of 2030 ± 504 U/mg. The restored ADA+ activity is stable over several months of continuous growth. It is identical to the activity of C57BL/6 thymic cells or C57BL/6 T tumor lines regarding (a) its sensitivity to ADA inhibitors 2‐deoxycoformycin and erythro‐9‐(2‐hydroxyl‐3‐nonyl)adenine, and (b) its electrophoretic mobility under nondenaturing conditions (starch gel and isoelectric focusing). An ADA‐specific, 1.4‐kb mRNA is present in the reactivated clones but is undetectable in the CTL ADA− parental line. These results demonstrate that the ADA− phenotype is due to an extinction of the corresponding gene. They suggest that the extinction of the ADA gene which appears to be specific for CTL and to take place in vivo during T cell differenciation may result from increased methylation in or near the ADA gene. This extinction seems to affect specifically ADA since nucleoside phosphorylase, the enzyme which follows ADA in the purine salvage pathway, is present at equivalent levels in the ADA− and ADA+ CTL clones.
Structural requirements for inducing in vitro B lymphocyte activation by chemically synthesized derivatives related to the nonreducing D‐glucosamine subunit of lipid A
Authors: Kumazawa Yoshio , Matsuura Motohiro , Maniyama Tokuei , Yuzuru Homma J. , Kiso Makoto , Hasegawa Akira
Published: February 2007

Mitogenic and polyclonal B cell activation (PBA) activities of 16 synthetic compounds related to the nonreducing D‐glucosamine (GlcN‐II) subunit of lipid A were investigated. Among compounds possessing the GlcN backbone, a 4‐O‐phosphorylated GlcN derivative carrying N‐linked 3‐tetradecanoyloxytetradecanoyl [C14‐O‐(C14)] and 3‐O‐linked tetradecanoyl (C14) groups, GLA‐27, expressed the highest degree of both activities. Omission of the 3‐O‐linked C14 group from GLA‐27 and transfer of the C14group to the C‐6 position induced critical changes in expression of activities. Both 4‐ O‐phosphorylated compounds carrying an N‐linked C14 or 3‐hydroxytetradecanoyl (C14OH) group instead of the C14‐O‐(C14) group in GLA‐27 showed no detectable activity. Substituting a 3‐O‐linked C14 group in GLA‐27 for the C14‐O‐(C14) group also markedly decreased mitogenic and PBA activities. Change of phosphorylation site from the C‐4 to the C‐6 position and bisphosphorylation at the C‐4 and C‐6 positions induced somewhat weak depression. Much weaker activities were observed in a compound carrying N‐linked 3‐dodecanoyloxydodecanoyl [C12‐O‐(C12)] and 3‐O‐ dodecanoyl (C12) as fatty acid substituents. No detectable activity was seen in a compound carrying N‐linked 3‐hexadecanoyloxyhexadecanoyl [C16‐O‐(C16)] and 3‐O‐ hexadecanoyl (C16), indicating that the most suitable carbon chain length for expressing the activities is C14. Regarding structural change of the GlcN backbone, a 1‐deoxy derivative of GLA‐27 exhibited stronger activity than did GLA‐27 itself. Mitogenic and PBA activity of GLA‐27 were stronger than those of lipid X, which corresponds to the reducing D‐GlcN (GlcN‐I) subunit of Escherichia coli lipid A and is a 1‐O‐phosphorylated GlcN derivative carrying N‐ and 3‐O‐linked C14OH groups. These results indicate that N‐linked acyloxyacyl and 3‐O‐linked acyl groups and phosphorylation are critical for expressing both mitogenic and PBA activities.
Ligand binding by the pl50,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3)
Authors: Malhotra Vivek , Hogg Nancy , Sim Robert B.
Published: February 2007

U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b‐binding proteins isolated by affinity chromatography on iC3b‐Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b‐Sepharose. The polypeptides of 170 and 95 kDa were identified as the α and the β subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150‐kDa polypeptide was not immunoprecipitated by antibodies to the α subunit of CR3 or LFA‐1. However, the 150‐kDa polypeptide, together with the 95‐kDa polypeptide, was immunoprecipitated with an anti‐β subunit‐specific antibody IB4, which immunoprecipitates LFA‐1, CR3 and pl50,95. These results indicated that the 150‐kDa polypeptide is the a subunit of the pl50,95 antigen. The binding of pl50,95 and CR3 to iC3b‐ Sepharose is specific as neither binds to C3u‐Sepharose.

A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95‐kDa polypeptide from U937 cells was characterized as being directed against the α‐subunit of the pl50,95 antigen. Phorbol myristate acetate‐stimulated U937 cells form rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for pl50,95 in adherence of iC3b‐coated particles. Since there is no rosetting with C3d‐bearing particles it is unlikely that pl50,95 is a receptor for C3d, a role for pl50,95 which has been suggested by others (Wright, S. D., Licht, M. R. and Silverstein, S. C, Fed. Proc. 1984. 43: 413).
Early arrest of B cell development in nude, X‐linked immune‐deficient mice
Authors: Karagogeos Domna , Rosenberg Naomi , Wortis Henry H.
Published: February 2007

Mice simultaneously expressing the nude and xid mutations have a severe deficit of both mature T and B cells. We now report studies designed to determine at which point in B cell differentiation development is arrested. Nude‐xid mice have normal numbers of hematopoietic colony forming units (CFU‐s) but lack two early pre‐B cell markers: susceptibility to transformation by Abelson murine leukemia virus (A‐MuLV) and production of cytoplasmic μ (Cμ) heavy chain. Thus, there is a defect in lymphocyte development prior to or early in pre‐B cell differentiation but after hematopoietic stem cell formation. The monoclonal reagents DNL 1.9, 14.8 and RA3–3A1/6.1, which react with the surface protein B220 (Ly 5) on pro‐B, Cμ− pre‐B, Cμ+ pre‐B, and surface Ig+ B cells, revealed the presence of positive cells in nude‐xid mice. The bone marrow of nude‐xid mice contains more B220+ cells than Cμ+ cells. Our data suggest that the developmental block in these mice occurs at the earliest identifiable step in the B lymphocyte lineage, after the appearance of B220+ Cμ− pro‐ B cells, but before the differentiation of Cμ‐bearing (pre‐B) cells.
Characterization of suppressor‐inducer cells which control the production of rat erythrocyte‐induced anti‐erythrocyte autoantibodies
Authors: Watt Graham J. , Elson Christopher J. , Healey Donald G. , Oryan Ahmad , Craig Hooper D.
Published: February 2007

Mice immunized with rat erythrocytes produce autoantibodies to their own red blood cells, distinct anti‐rat agglutinins and autoantigen‐specific suppressor cells. Suppressor cells were detected by adoptive transfer of rat erythrocyte‐immunized spleen cells to naive recipients. Such recipients failed to make erythrocyte autoantibodies after immunization with rat erythrocytes although their anti‐rat erythrocyte response was unimpaired. Depletion and enrichment studies were performed to identify the cell type(s) which transfer suppression. B cell depletion of rat erythrocyte‐immunized spleen cells by passage over Ig/anti‐Ig‐coated bead columns abrogated the transfer of suppression. However, suppression was still transferred after rat erythrocyte immunized spleen cells were passed over beads coated with a complex of 4‐azido‐2‐ nitrophenyl (NAP)‐mouse IgG‐rabbit IgG anti‐NAP suggesting that T cells bearing Fcγ receptors are not responsible for suppression. Positively selected B cells from rat erythrocyte‐immunized spleen cells caused some suppression of erythrocyte autoantibodies but only after high numbers of cells were transferred. Neither positively selected Lyt‐l+2− nor Lyt‐1–2+ T cell subpopulations transferred suppression. By contrast, rat erythrocyte‐immunized spleen cells which contained a mixture of B memory and T cells were suppressive and retained their suppressor activity after removal of Lyt‐1–2+ but not Lyt‐l+2− cells. It is proposed that these Lyt‐l+2− T cells belong to a distinct population of suppressor‐inducer cells which together with memory B cells stimulate the generation of effector T suppressor cells in naive recipients.
Biosynthesis of the subcomponents Clq, Clr and Cls of the first component of complement (Cl) by guinea pig hepatocyte primary cultures
Authors: Ramadori Giuliano , Heinz Hans‐Peter , Martin Harry , Buschenfelde Karl‐Hermann Meyer Zum , Loos Michael
Published: February 2007

Thus far, the synthesis of Clq by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (106 cells/ml) were cultured under serumfree conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4–8) antibody directed against a nonpolymorphic la determinant and preabsorbed rabbit serum. The hemolytic activity of Cl and its subcomponents Clq and Clr/Cls was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 × 103 effective Clq molecules/cell/24 h and 34 ± 103effective Clr/Cls molecules/cell/24 h. The synthesis of Clq and Clr/Cls could be reversibly inhibited by cycloheximide (50 μg/ml). Furthermore, to demonstrate de novo synthesis of the Clq subcomponent, endogeneous labeling with 3H‐proline (or 14C‐proline) was performed. The immunoprecipitated Clq from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig Clq from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize Cl subcomponents, whereby the synthesis of Clq and Clr/Cls occurs independently.
In vivo suppression of perinatal multispecific B cells results in a distortion of the adult B cell repertoire
Authors: Vakil Meenal , Sauter Helmut , Paige Christopher , Kearney John F.
Published: February 2007

The isolation of multispecific B cell hybridomas with a variety of anti‐idiotype (anti‐ Id) activities from the lymphoid organs of fetal and neonatal B ALB/c mice suggested that the development of the immune system may depend on Id interactions among autologous B cells. In vitro analysis of antibodies secreted by these hybridomas showed extensive sharing of an idiotope defined by the monoclonal antibody FD5–1. Early and timed administration of this antibody during the perinatal period results in a distortion of the phosphorylcholine (PC) and a(l±3)dextran (Dex)‐specific B cell precursor compartment of the developing repertoire and is reflected by a drastic reduction of antibody responses to these antigens when challenged as adults. These observations provide strong evidence for the involvement of the early appearing multispecific B cells in Id interactions that bring about the uniform development of the normal adult B cell repertoire. Interference with these interactions at critical stages of development results in permanent deficiencies in the adult B cell repertoire.
Epstein‐Barr virus‐induced IgE production in limiting dilution cultures of normal human B cells
Authors: Stein Leonard D. , Chan Marcia A. , Hibi Toshifumi , Dosch Hans‐Michael
Published: February 2007

The induction of in vitro IgE production in human B cells from normal, nonatopic donors has been difficult and somewhat controversial. We report that IgE production is consistently observed in limiting dilution cultures of in vitro Epstein‐Barr virus (EBV)‐infected normal human B lymphocytes. The frequency of IgE‐committed, EBV‐responsive cells ranged from 1/810 to 1/10000 B lymphocytes, and it was similar in peripheral (blood, tonsil) and central (bone marrow) tissue sites. Poisson distribution analysis of these limiting dilution cultures suggested that IgE‐committed B cells comprise 0.1–1% of all EBV‐responsive B lymphocytes.
In vivo administration of interleukin 2 stimulates mitosis in thymus and bone marrow
Authors: Gearing Andrew J. H. , Wadhwa Meenu , Perris Alan D.
Published: February 2007

We have used short‐term, high‐density cultures to demonstrate that interleukin 2 (IL 2) in picomolar amounts causes entry of approximately 2% of thymocytes from 3‐ month‐old rats into mitosis. Newborn and fetal animals show a higher response reflecting a greater proportion of cells which have been shown to express IL 2 receptors at this age. In vivo administration of nanogrammes of IL 2 or injection of rats with syngeneic spleen cells which had been stimulated in vitro with concanavalin A to release IL 2 were also shown to increase the proliferation of both thymus and bone marrow cells. This suggests that IL 2, in amounts which could be produced by peripheral lymphoid tissue during immune responses, could act to increase the turn‐over of lymphocytes in bone marrow and thymus.
Does the deletion within T cell receptor β‐chain gene of NZW mice contribute to autoimmunity in (NZB × NZW)F1mice?
Authors: Yanagi Yusuke , Hirose Sachiko , Nagasawa Ryuji , Shirai Toshikazu , Mak Tak W. , Tada Tomio
Published: February 2007

To determine the transacting genetic factors of NZW contributing to the development of autoimmune disease in (NZB ± NZW)F1 (B/WF1) mice, we examined the relationship between the T cell receptor β chain gene deletion and the severity of autoimmune manifestations in 76 B/WF1 ± NZB backcross mice. Very high association between the T cell receptor β chain gene deletion and the development of autoimmune manifestations including the production of IgG anti‐DNA antibodies and circulating retroviral gp70 immune complexes was observed, indicating that a defect in the NZW T cell receptor β chain gene or a locus closely linked to it contributes to the autoantibody formation in B/WF1.
Insulin‐specific T cell help to TNP‐specific B cells requires activation via the antigen T cell receptor
Authors: Rubin Bent , Boned Annie
Published: February 2007

In the present study, the production of large numbers of insulin (Ins)‐specific, H‐2‐ restricted T helper (Th) cell clones is described. Among 148 clones analyzed, 121 clones had Th cell function, and these were divided into 75 Ins‐specific Th cell clones and 52 autoreactive clones. The 148 clones were isolated from Ins‐specific T cell lines produced by in vitro stimulation of T lymphocytes from (b ± d)F1 mice immunized with Ins 7, 14, 32 or 56 days before. The following characteristics were tested with regard to the Th, cell clones: restriction specificity and antigen requirement for optimal help or interleukin 2 (IL2) production. No differences in these characteristics were found among clones originating from day 7, 14, 32 or 56 T cell lines. A preference for H‐2b as restriction element and an antigen concentration of about 0.01 μg trinitrophenylated (TNP)‐Ins/ml for optimal help were general traits. Optimal IL2 release is not yet obtained with 100 ug TNP‐Ins/ml. Thus, the antigen requirement for optimal help and IL 2 release differs by a factor 104 at least.

Certain twice‐cloned Th cell lines were tested for IL2 production when stimulated with anti‐Thy‐1 monoclonal antibodies (mAb). All clones analyzed were stimulated by mAb H155 but not with mAb H140 nor with mAb against Lyt‐1, L3T4, LFA‐1 or H‐2K/D molecules. Therefore, we determined whether TNP‐conjugated H155 mAb would mediate Th cell‐B cell collaboration as well as TNP‐Ins. The results with nine different Th cell clones and six different TNP‐conjugated mAb used in a 107‐fold concentration range showed that Th cell clones have to be triggered via the T cell receptor for expression of helper function to B cells.

Thus, though IL2 gene activation, synthesis or release apparently can be activated via at least two pathways: (a) T cell receptor or (b) Thy‐1, it seems that activation of the genes responsible for synthesis and release of the helper factors, which ensure antigen‐specific B cell proliferation and differentiation, needs Th cell‐B cell contact mediated via the antigen‐specific T cell receptor.
B cell hyperactivity in systemic lupus erythematosus: selectively enhanced responsiveness to a high molecular weight B cell growth factor
Authors: Delfraissy Jean‐François , Wallon Christine , Vazquez Aimé , Dugas Bernard , Dormont Jean , Galanaud Pierre
Published: February 2007

To characterize B cell hyperactivity in systemic lupus erythematosus (SLE) patients we studied the early events of B cell activation in 14 patients and controls. We measured B cell proliferation induced by three interleukin (IL) preparations (20‐kDa B cell growth factor, BCGF, recombinant IL2 and 50‐kDa BCGF) in the absence and in the presence of an anti‐μ antibody (Ab). SLE B cells exhibited a markedly enhanced proliferative response to the 50‐kDa BCGF in the absence of an anti‐μ Ab, while responding normally in the presence of a first signal. This pattern of hyperactivity was observed in 11 out of 14 patients tested, and was absent in control patients. In contrast, SLE B cells behaved like normal B cells for the response to the other two IL tested, and to the anti‐μ Ab alone. It should be pointed out that SLE B cells responded normally to recombinant IL2 whereas T cells from the same patients exhibited a decreased response to this IL. The selectively enhanced responsiveness of SLE B cells to the 50‐kDa BCGF suggests that the events leading to B cell hyperactivity in this disease affect the early stages of B cell activation.
Immunoglobulin‐bearing B cells reconstitute and maintain the murine Ly‐1 B cell lineage
Authors: Hayakawa Kyoko , Hardy Richard R. , Stall Alan M. , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: February 2007

Previous reconstitution studies in irradiated mice distinguished Ly‐1 B from other B cells. The predominant B cell populations in spleen and lymph node are readily reconstituted from progenitors present in adult bone marrow. Ly‐1 B cells, in contrast, are reconstituted from cells in peritoneum rather than bone marrow. Further studies, presented here, demonstrate that Ly‐1 B populations are reconstituted from peritoneal cells that already express both surface immunoglobulin (IgM) and Ly‐1, that is, Ly‐1 B. Such cells reconstitute and maintain the Ly‐1 B population in irradiated recipients for at least 6 months.
VLA‐3: A novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization
Authors: Sánchez‐Madrid Francisco , De Landázuri Manuel O. , Morago Gloria , Cebrián Marta , Acevedo Augustín , Bernabeu Carmelo
Published: February 2007

The family of human T cell activation‐associated proteins, named VLA complex, is formed by the molecular association of cell surface glycoproteins of 210, 165, 130 and 80 kDa. In this report, we describe eight different monoclonal antibodies (HP mAb) specific for the 80‐kDa polypeptide which preferentially associates with the 165‐kDa member. Comparative immunoprecipitation and cell‐binding studies demonstrated that the HP mAb recognize an epitope(s) on the 165/80 kDa subset different from those recognized by other anti‐VLA mAb previously described. Furthermore, cellular and tissue distribution studies by flow cytometry and peroxidase staining showed that the HP reactivity pattern is different from other VLA specificities. The 165/80‐kDa complex defined by HP mAb is present on human thymocytes, peripheral blood lymphocytes as well as on T, B and myelomonocytic cell lines. However, only the 80‐kDa subunit was precipitated by HP mAb from activated T lymphocytes. These results suggest that the association between the 165‐ and 80‐kDa subunits diminishes during the activation process, and that the epitopes recognized by the HP mAb are located on the 80‐kDa protein. The novel polypeptide association of 165/80 kDa has been termed VLA‐3.
The molecular evolution of the immune response: idiotope‐specific suppression indicates that B cells express germ‐line‐encoded V genes prior to antigenic stimulation
Authors: Manser Tim , Gefter Malcolm L.
Published: February 2007

Antibodies expressed by the immune B cell population are characterized by variable region amino acid substitutions resulting from somatic nucleotide replacement (somatic mutation). This is not true of antibodies expressed by the “naive” B cell population. It is at present unclear whether this discrepancy is due to the preferential clonal selection of a pre‐existing subpopulation of naive B cells that express variable regions altered via nucleotide replacement, or whether the process of nucleotide replacement occurs only during the antigen‐dependent stages of B cell differentiation. To address this question we have used anti‐idiotypic suppression to functionally delete B cells that express particular variable‐region structures from the antigen‐responsive repertoire. Suppression of the major cross‐reactive idiotype (IdCR) expressed in strain A mice in response to p‐azophenylarsonate (Ars) was induced using the monoclonal anti‐IdCR antibody AD8. The idiotope recognized by AD8 is easily destroyed by alteration of IdCR variable‐region structure via nucleotide replacement. The IdCR anti‐Ars immune repertoire is characterized by antibodies that lack the AD8‐cognate idiotope due to nucleotide replacement. However, complete suppression of the IdCR could reproducibly be achieved by administration of AD8 prior to Ars immunization. This result indicates that all IdCR‐expressing B cells also express the AD8‐cognate idiotope prior to immunization. Thus, somatic nucleotide replacement must occur exclusively during the antigen‐dependent stages of B cell differentiation in this system.
Phenotypic characterization of individual interferon‐γ‐producing cells after OKT3 antibody activation
Authors: Andersson Ulf , Laskay Tamas , Andersson Jan , Kiessling Rolf , Deley Marc
Published: February 2007

This is the first study using a direct approach to determine the phenotype of interferon gamma (IFN‐γ)‐producing cells after polyclonal T cell activation. Blood mononuclear cells (MNC) were cultured and stimulated by OKT3 antibody to produce IFN‐γ. They were then stained with a panel of monoclonal antibodies with specificity for cell surface antigens, fixed and permeabilized in suspension and stained for cytoplasmic IFN‐γ using monoclonal antibodies and indirect immunofluorescence technique. IFN‐γ appeared mainly as a local, polar accumulation in the cytoplasm in a juxtanuclear position, probably identical to the Golgi apparatus. After a culture period of 20 h, 6–11% and 0.2–0.6% of the OKT3‐activated MNC from adults or newborns, respectively, produced IFN‐γ in cultures. Unstimulated cultures contained maximally 0.1% IFN‐γ‐positive MNC. The majority of the IFN‐γ‐producing MNC were T cells and expressed the T11 antigen and receptors for interleukin 2. To our surprise most of these T cells also carried receptors for complement 3bi (OKM1) but did not express class II molecules. Less than 50% displayed T4 or T8 antigens in three out of the four experiments performed. Natural killer cells, as recognized by Leu7 or B73.1 antibodies, contributed only marginally to the IFN‐γ synthesis whereas B cells (OKB7) and monocytes (Leu M3) showed no production of IFN‐γ.