bluuebunny/crossref_metadata_2025 huggingface dataset:
The molecular context of determinants within the priming antigen establishes a hierarchy of T cell induction: T cell specificities induced by peptides of β‐galactosidase vs. the whole antigen
Authors: Shivakumar Sumati , Sercarz Eli E. , Krzych Urszula
Published: March 2007

The antibody response following priming with a macromolecule or a peptide will depend on the regulatory T cells that become activated by the antigenic determinants available. In this report, activation of T helper (Th) and T suppressor (Ts) cells by determinants on β‐galactosidase (GZ) was examined by comparing native GZ [1023 amino acid (a.a.) residues per monomer] with peptides from the immunodominant region encompassing residues 3 to 187. Each immunogen established its characteristic hierarchy of dominance of determinants within it: in particular, GZ and CB‐2–3 (a. a. 3–187) each induced immunodominant Th cells which could not be induced by T8 (a.a. 60–140). Hierarchies of suppressor determinant are also created: T8‐Ts suppresses all Th specificities and therefore can be deemed immunodominant: T8–2‐Ts and T8–3‐Ts have a more selective suppressor activity and can be considered subdominant. We conclude that the outcome of immunization shifts with a change in the nature of the immunogen and the context within which the determinant lies will crucially influence its expression. A particular ‘context’ presumably determines the likely order of processing of that molecule which leads to a characteristic relationship among the Ts, Th and B cell determinants involved.
On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases
Authors: Frei Karl , Malipiero Ursula V. , Leist Thomas P. , Zinkernagel Rolf M. , Schwab Martin E. , Fontana Adriano
Published: March 2007

Interleukin 6 (IL 6) was found to be produced in the central nervous system (CNS) of ICR +/+ mice infected with lymphocytic choriomeningitis virus (LCMV) or with vesicular stomatitis virus (VSV). When infecting athymic ICR nu/nu mice which cannot develop T cell‐mediated meningitis after LCMV infection, no significant synthesis of IL 6 was detected in the CNS. IL 6 was found, however, to be produced intrathecally in ICR nu/nu mice infected with VSV, which causes a T cell‐independent acute encephalitis. This suggested that IL 6 may also originate from cells not belonging to the T cell compartment. Indeed, in vitro assays showed that both virus‐infected microglial cells and astrocytes secreted IL 6. In astrocytes, the infection resulted in the induction of the 1.3‐kb messenger RNA IL 6. Besides its effect on the development of B cell immunity in the brain, IL 6 may be involved in repair mechanisms initiated in the course of viral‐induced tissue damage. As shown here, IL 6 induced an increase of the secretion of a neurotrophic factor, nerve growth factor by astrocytes. Thus, the intrathecal synthesis of IL 6 may be part of the host response to infection favoring immune‐mediated elimination of the infectious agent as well as trophic support for neurons.
The role of anti‐idiotypic antibodies in the induction of experimental systemic lupus erythematosus in mice
Authors: Mendlovic Shlomo , Fricke Harald , Shoenfeld Yehuda , Mozes Edna
Published: March 2007

We have recently reported the induction of experimental systemic lupus erythematosus (SLE) in mice by a human anti‐DNA monoclonal antibody (mAb) that bears a common idiotype, the 16/6 Id. In the present report we investigated the role of the idiotypic network in the induction of experimental SLE by using a murine anti‐idiotypic mAb specific for the 16/6 Id. This anti‐idiotypic mAb induced experimental SLE similarly to the 16/6 Id. Thus, following immunization, in addition to 16/6 Id+ antibodies, the mice produced antibodies to various nuclear antigens: single‐stranded DNA, double‐stranded DNA, poly(I), poly(G), Ro, La, Sm and ribonucleoproteins. Similarly to the 16/6 Id‐immunized mice, the mice injected with the anti‐16/6 Id mAb exhibited elevated erythrocyte sedimentation rate and leukopenia. The murine anti‐16/6 Id mAb was found to be more effective than the 16/6 Id, in causing earlier onset of proteinuria and renal damage. These results suggest that the idiotypic network and particularly anti‐idiotypic antibodies specific for anti‐DNA common idiotypes found in SLE, play an important role in the induction of SLE in mice.
Interleukin (IL) 4 counteracts the helper effect of IL 2 on antigen‐activated human B cells
Authors: Llorente Luis , Crevon Marie‐Claude , Karray Saoussen , Defrance Thierry , Banchereau Jacques , Galanaud Pierre
Published: March 2007

We tested the effect of interleukin (IL)4 on the specific IgM antibody response induced by trinitrophenylated‐polyacrylamide beads (TNP‐PAA) in cultures of human B cells. T cell help was provided by exogeneous IL2. IL4 profoundly suppressed the response to optimal concentrations (50 U/ml) of IL2, with a 50% inhibitory concentration of 6 U/ml. This was due neither to a shift in the kinetics nor to a switch to an IgG response. The production of anti‐TNP antibody (as measured by an enzyme‐linked immunosorbent assay in the culture supernatant) was inhibited to the same extent as the generation of plaque‐forming cells. The effect of IL4 was completely abolished by a neutralizing antibody toward IL4. Kinetic studies showed that IL4 had to be present during the first 48 h of culture to fully inhibit the response. The sequential stimulation of B cells by antigen and by IL2 showed that IL4 does not negatively interfere with signaling through membrane Ig but counteracts the effect of IL2 on antigen‐activated B cells.
Membrane anchoring and spontaneous release of CD16 (FcR III) by natural killer cells and granulocytes
Authors: Lanier Lewis L. , Phillips Joseph H. , Testi Roberto
Published: March 2007

CD16 is an IgG Fc receptor that is predominantly expressed on human natural killer (NK) cells and granulocytes. The CD16 antigen expressed on neutrophils is a 50 to 70‐kDa glycoprotein attached to the plasma membrane by a phosphatidylinositol‐glycan linkage that is susceptible to cleavage by phosphatidylinositol‐specific phospholipase C (PIPLC). By contrast, treatment of NK cells with PIPLC did not cleave CD16 from the cell surface. Biochemical analysis of the deglycosylated CD16 revealed a substantial difference in the core polypeptides expressed by NK cells and granulocytes. Culture of radiolabeled NK cells resulted in the spontaneous release of a CD16 molecule, smaller than the membrane form of CD16. These findings demonstrate that structural differences exist in the CD16 antigens expressed on NK cells and granulocytes that may potentially influence their functional activities.
Resistance of fetal PVG rats to induction of allograft tolerance
Authors: Mccullagh Peter
Published: March 2007

Artificially induced immunological tolerance is often envisaged as experimental mimicry of events which occur naturally during the acquisition of self tolerance. Thus, it has been inferred that similar circumstances will facilitate the induction of both types of unresponsiveness. Since tolerance to most self determinants appears to have been established by the time of birth and allograft tolerance can be experimentally induced more readily, if not exclusively, in very young animals, it is commonly assumed that susceptibility to induction of tolerance to foreign antigens will continue to increase as progressively younger animals are tested. The experiments reported in this study extend the range of circumstances under which younger animals may be found to be less susceptible, or even completely resistant, to tolerance induction.

While neonatal PVG rats were highly susceptible to the induction of tolerance to (PVG × DA)F1 hybrid determinants, fetal PVG rats were only partially susceptible at 19 days and were completely resistant at 17 and 18 days of gestation. Furthermore, only 39% of neonatal PVG rats that had been inoculated with F1 hybrid bone marrow cells at 18 days of gestation could be rendered tolerant by procedures which were effective in 100% of previously untreated neonates. These observations were interpreted to indicate that a certain stage of maturity must be attained before fetal rats become susceptible to tolerance induction.

Asymmetry in susceptibility to tolerance induction in response to the inoculation of F1 hybrid bone marrow cells was observed when PVG and DA fetuses, but not neonates, were compared. This difference in the response of the two strains recalls the divergence in immune reactivity of offspring, previously reported when PVG and DA embryos were transferred to surrogate mothers of the other strain. It is also in agreement with earlier reports of substantial differences in the ease of tolerance induction between different strain combinations. The asymmetry between PVG and DA rats in the present experiments is speculatively attributed to differences in rates of immunological maturation of the two strains.
Enhanced responsiveness of human memory T cells to CD2 and CD3 receptor‐mediated activation
Authors: Sanders Martin E. , Makgoba Malegapuru W. , June Carl H. , Young Howard A. , Shaw Stephen
Published: March 2007

Previous investigations have defined phenotypic differences between unprimed (naive) and antigen‐primed (memory) T cells from human peripheral blood. We now report that memory T cells proliferate much more than naive cells when stimulated with anti‐CD3 monoclonal antibody or pairs of anti‐CD2 monoclonal antibodies. Enhanced responsiveness to receptor‐mediated triggering is a novel mechanism for T cells which could facilitate memory cell response to specific antigen. Furthermore, when triggered via either CD2 or CD3, memory T cells produce substantial amounts of interferon gamma while naive cells produce virtually none; this suggests that differentiation from naive to memory state is accompanied by a stable change in regulation of the gene for interferon‐γ. We conclude that naive and memory T cells are dramatically different in function as well as phenotype.
Expression and function of AIM, an activation inducer molecule of human lymphocytes, is dependent on the activation of protein kinase C
Authors: Cebrián Marta , Redondo Juan Miguel , López‐Rivas Abelardo , Rodríguez‐Tarduchy Gemma , De Landázuri Manuel O. , Sánchez‐Madrid Francisco
Published: March 2007

AIM is an activation inducer molecule selectively expressed by activated lymphocytes through which agonistic proliferative signals can be triggered. The relationship between the expression of AIM with the activation of protein kinase C (PKC) has been studied. Different activators of PKC such as the active phorbol esters, phorbol myristate acetate and phorbol dibutyrate, or the phorbol‐related ester mezerein were able to induce AIM expression on peripheral blood lymphocytes as assessed by immunofluorescence flow cytometry. Moreover, the expression of this activation antigen was also induced by treatment of peripheral blood lymphocytes either with dioctanoyl‐rac‐glycerol, a synthetic analogue of diacylglycerol, the physiological mediator of PKC activation. Further indirect evidence that AIM expression was dependent on the activation of PKC was obtained by blockade of the induction of its expression in cells treated with H7, an inhibitor of PKC. The AIM expression can be detected as early as 3 h after addition of phorbol esters and it requires active RNA and protein synthesis. The activation of PKC appears to be also required in the proliferative response induced by anti‐AIM monoclonal antibody (mAb) in conjunction with phorbol esters. Agents such as phorbol myristate acetate, phorbol dibutyrate or mezerein but not the inactive phorbol ester methyl‐phorbol myristate acetate induced a high proliferation of peripheral blood lymphocytes in the presence of anti‐AIM mAb. In addition, we have demonstrated that the anti‐AIM mAb is not sufficient by itself to induce cellular proliferation once the AIM antigen is expressed at the cell surface, requiring the simultaneous stimulation of the PKC to trigger high proliferative responses. Furthermore, the anti‐AIM mAb did not appear to exert its effect on proliferation by rapidly increasing the intracytoplasmic Ca2+ levels. Taken together all these results indicate that the expression and function of AIM antigen is dependent on the activation of PKC.
Dual role of macrophages in the suppression of interleukin 2 production and interleukin 2 receptor expression in trypanosome‐infected mice
Authors: Sileghem Maarten , Darji Ayub , Hamers Raymond , Winkel Marnix Van De , Baetselier Patrick De
Published: March 2007

Lymph node cells derived from T. brucei‐infected mice fail to produce interleukin 2 (IL 2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL 2 in co‐culture experiments. The depletion of Thy‐1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell‐enriched nylon wool‐nonadherent fraction, on the other hand, is not suppressive. The suppression of IL 2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin‐producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather directly interfere with the normal lymph node cells.

In contrast to the essential role of prostaglandins in the impairment of IL 2 production, these mediators are not involved in the suppression of IL 2 receptor expression. Lymph node cells derived from Trypanosoma brucei‐infected mice fail to produce interleukin 2 (IL 2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL 2 in co‐culture experiments. The depletion of Thy‐1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell‐enriched nylon wool‐nonadherent fraction, on the other hand, is not suppressive. The suppression of IL 2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin‐producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather interfere directly with the normal lymph node cells.
Developmentally regulated expression of the neural cell adhesion molecule (NCAM) by mouse thymocytes
Authors: Brunet Jean‐François , Hirsch Marie‐Rose , Naquet Philippe , Überla Klaus , Diamantstein Tibor , Lipinski Marc , Goridis Christo
Published: March 2007

The expression of the neural cell adhesion molecule (NCAM) has been investigated during thymus ontogeny. NCAM mRNA was readily detectable at day 19 of gestation, the youngest age studied. Its level declined after birth to become undetectable at 3 weeks of age. Cell surface expression of NCAM protein was detected on 14% of day 15 fetal thymocytes and peaked during the perinatal period, when around 40% of the thymocytes expressed low to medium levels of NCAM. At postnatal day 2, the vast majority of the NCAM+ cells were also CD4+ and CD8+. At embryonic day 15, NCAM appeared also to be expressed by CD4− thymocytes since 14% of the cells were already NCAM+ whereas CD4 was virtually undetectable. In frozen section of the newborn thymus, surface staining for NCAM was present on a subpopulation of cells in the cortex, rare in the medulla and absent from the sub‐capsular area. In conjunction with other cell adhesion molecules, NCAM could play a role in cell interactions during thymic development.
Translation affects immunoglobulin mRNA stability
Authors: Jäck Hans‐Martin , Berg Jörg , Wabl Matthias
Published: March 2007

When termination codons were introduced into exons of the gene for Ig m̈ chain, steady‐state levels of m̈ mRNA were reduced, both at the pre‐B cell stage and at the plasma cell stage. A termination codon in the variable region gene segment and a termination codon in the second exon of the constant region gene segment had effects of similar magnitude. When the termination codon was deleted, the original level of mRNA was restored. The rate of m̈ gene transcription was the same whether or not a termination codon was present. Therefore, the termination codons must reduce the amount of the mRNA by reducing its stability. Since the introduced termination codons prematurely terminate translation and, in so doing, change the ribosome load on the mRNA, we conclude that m̈ mRNA stability is conferred in part by ribosomal protection from enzymatic degradation. We propose that the differences in m̈ mRNA stability during B lymphocyte differentiation are due to different amounts of ribosomes available for translation.
The effects of anti‐CD2 antibodies on the differentiation of mouse thymocytes
Authors: Kyewski Bruno A. , Jenkinson Eric J. , Kingston Rosetta , Altevogt Peter , Owen Michael J. , Owen John J. T.
Published: March 2007

Using a rat monoclonal antibody against mouse CD2, we determined the expression of this marker on thymocytes during ontogeny. CD2 expression becomes detectable at day 15 and reaches adult levels (∼95% positivity) by day 19. Furthermore, the effect of anti‐CD2 antibodies on T cell differentiation was analyzed by addition of antibodies to thymus organ cultures or repeated injection into newborn mice. Anti‐CD2 antibodies inhibit CD2 expression in organ cultures and drastically reduce its expression on thymocytes and peripheral lymphocytes in vivo. In either situation, suppression of CD2 expression does not significantly alter the generation of T cells expressing CD3, CD4, CD8 and T cell receptor Vβ8. These results do not support a role for CD2 in early steps of thymocyte differentiation.
E‐series prostaglandins are potent growth inhibitors for some B lymphomas
Authors: Phipps Richard P. , Lee David , Schad Victoria , Warner Garvin L.
Published: March 2007

The ability of prostaglandins (PG) to inhibit the growth of B cell lymphomas was investigated. Macrophage‐secreted PGE2 was previously shown to promote unresponsiveness to antigen in normal B lymphocytes. This observation suggested that B lymphomas might also be regulated by prostanoids. Five non‐PG‐secreting Ly‐1+ B lymphomas (CH12, CH31, CH33, NBL and WEHI‐231) were incubated for 24–72 h with PGE2, PGE1 or PGF2 α. The level of lymphoma growth at the end of culture was determined using a colorimetric assay which detects only viable cells. A marked heterogeneity was observed with respect to the sensitivity of these lymphomas to PGE2 and PGE1. CH31 was very sensitive, being growth inhibited by as little as 10−8 M PGE. In contrast, CH12, a more mature lymphoma, was highly resistant, whereas CH33, NBL and WEHI‐231 were of intermediate resistance. All five lymphomas demonstrated little or no growth inhibition when cultured with PGF2 α. Moreover, unlike PGE2, PGF2 α failed to elevate intracellular cAMP levels. It was previously shown that CH31, CH33 and WEHI‐231 could be growth inhibited by anti‐immunoglobulin antibodies which cross‐link surface immunoglobulin. Interestingly, these three lymphomas were rendered more sensitive to this treatment if PGE2 was present. For example, 10−8 M PGE2 alone had little effect on CH33, but significantly augmented growth inhibition induced by suboptimal quantities of anti‐immunoglobulin antibody. Cholera toxin, another agent which was found to rapidly elevate intracellular cAMP levels, also synergized with suboptimal doses of anti‐immunoglobulin to induce growth inhibition. Overall these data suggest that, in vivo, macrophage‐secreted prostanoids may slow the growth of some lymphomas and that anti‐immunoglobulin or anti‐idiotype treatment may be more effective in the presence of agents which elevate cAMP such as E‐series PG.
Mapping of murine IgE epitopes involved in IgE‐Fcε receptor interactions
Authors: Schwarzbaum Shelley , Nissim Ahuva , Alkalay Irit , Ghozi Mireille C. , Schindler Daniel G. , Bergman Yehudit , Eshhar Zelig
Published: March 2007

The generation of anti‐IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (FcER) interaction site has been determined using cross‐inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the FcεR interaction site and the binding sites of various anti‐IgE mAb, we employed site‐directed mutagenesis of the IgE heavy chain gene.

To this end we have constructed and expressed a recombinant murine constant E heavy chain (Cε) gene bearing a (4‐hydroxy‐3‐nitrophenyl)acetic acid (NP)‐binding VH region. Several site‐specific mutants in the Cε3 and Cε4 domains of this recombinant Cε gene were prepared and expressed by transfection into the light chain‐producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti‐IgE mAb.

The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the Cε3 domain, a mutant with a truncated Cε4 domain, a mutant with a 45 amino acid deletion in the carboxy end of Cε3, and a chimeric human Cε in which the human Cε3 was replaced by the homologous mouse Cε3 domain. These mutants have permitted the localization, to the Cε3 domain, of the epitopes recognized by the 84.1C and 95.3 anti‐IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the FcεR binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the FcεR binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE‐FcεR interaction, has been mapped to the Cε4 domain. When tested for binding to the FcεR on RBL‐2H3 cells, the point mutant bound to the FcεR with twofold reduced affinity, while the Cε3 deletion mutant and the mutant truncated in Cε4 lost all receptor binding activity. These data suggest that the FcεR binding site can be assigned to the third C region domain, and that the fourth domain, while not directly involved in FcεR binding, may play a role in the formation of the H2L2 tetrameric IgE molecule, and in stabilizing the conformation of IgE required for FcεR binding.
Cultured keratinocyte grafts are recognized, but not rejected by CD8+ T cells in vivo
Authors: Tinois Estelle , Cobbold Steve , Faure Michel , Yeoman Helen , Stanley Margaret
Published: March 2007

It has been shown in the mouse that cultured keratinocyte allografts [fully major histocompatibility complex (MHC) mismatched] survive for at least 100 days without evidence of rejection. In an attempt to analyze the immune mechanisms underlying this phenomenon we have investigated the induction of tolerance to such grafts. Primary cultures of BALB/c keratinocytes were prepared using irradiated 3T3 feeder cells, and the cultured cell sheets were grafted, using a silicone transplantation chamber, onto CBA recipients. After the cultured grafts had been in place for 4–6 weeks full‐thickness tail skin from BALB/c donors was grafted onto the dorsal flank opposite the cultured graft. The median graft survival time of these full‐thickness allografts was 15.5 days compared to 13 days in the control group.

These data show that in the absence of Langerhans cells and MHC class II expression, keratinocytes expressing class I and minor histocompatibility antigens induce a prolonged survival of full‐thickness skin allografts. The results from experiments in which T cell subsets were depleted in vivo suggest that CD8+ cells (a) recognize the class I alloantigens of the cultured graft, (b) do not reject the cultured graft and (c) do not progress further in their maturation pathway. We propose that these CD8+ T cells might have been partially primed by receiving only the first signal of activation and that they may be equivalent to ‘poised’ cytotoxic T cells. These CD8+ cells can reject non cultured full‐thickness skin grafts, and do so more effectively after removing CD4+ cells.
Cytokine mRNA levels in antigen‐stimulated peripheral blood mononuclear cells
Authors: Gauchat Jean‐François , Gauchat Dominique , De Weck Alain L. , Stadler Beda M.
Published: March 2007

Levels of cytokine mRNA coding for granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interferon‐γ (IFN‐γ), interleukin (IL) 2, IL 1β, IL 4 or IL 6 have been measured by Northern blot analysis after antigen stimulation. As source for RNA we used peripheral blood mononuclear cells (PBMC) from donors which showed a proliferative response after tetanus toxoid or Candida albicans stimulation. For comparison PBMC were also stimulated with lectins and anti‐CD3 antibody. With some variations among donors, antigens clearly induced measurable levels of IFN‐γ, GM‐CSF and IL 2 mRNA. Increased levels for IL 6 were also detected after antigen stimulation. In contrast to polyclonal T cell stimuli, antigens showed delayed kinetics of mRNA steady‐state levels and resembled in this respect more closely the stimulation with pokeweed mitogen. Thus, cytokine mRNA levels may be assessed in unfractioned PBMC after antigen stimulation. The two tested antigens also clearly show a cytokine pattern distinct from that induced in polyclonal stimulations such as anti‐CD3.
Two monoclonal antibodies specific for the T cell receptor Vα8
Authors: Tomonari Kyuhei , Lovering Erin , Fairchild Sue , Spencer Sarah
Published: March 2007

Two monoclonal antibodies, KT50 and KT65, specific for Vα8 have been established. This was determined as follows: (a) 4 T cell clones, C6, R1, G22 and 19, out of 43 T cell clones with various antigen specificities, major histocompatibility complex restrictions and Vβ usages not only bound KT50 and KT65 but also expressed Vα8 mRNA, (b) KT50 and KT65 precipitated molecules from the clone C6 similar to the T cell receptor molecules precipitated in C6 cells by KT11 (anti‐Vβ11) or KTL2 (anti‐Ti) and (c) KT50 and KT65 were mitogenic and induced cytotoxicity. All strains of mice so far examined have populations of KT50+ and KT65+ T cells of 1.4%−3.6% and 0.9%−2.6%, respectively. Different H‐2 haplotypes were not observed to affect the number of cells expressing KT50 or KT65. In addition KT15 (anti‐CD8), without cross‐linking to KT50 or KT65, augmented proliferation triggered by KT50 or KT65.
Effect of cyclosporin A on T cell immunity II. Defective thymic education of CD4 T helper cell function in cyclosporin A‐treated mice
Authors: Fukuzawa Masahiro , Sharrow Susan O. , Shearer Gene M.
Published: March 2007

Cyclosporin A (CsA) is an immunosuppressive agent that is widely used in transplantation. Recent animal studies indicate that CsA can affect the development of immunity so that autoreactive T lymphocytes are generated. In this study, mice were treated with CsA prior to irradiation and transplantation of syngeneic bone marrow to determine whether CsA pretreatment would affect the ability of the bone marrow recipients to develop normal T cell function. Our results indicate that (a) thymuses of CsA‐treated mice do not contain single‐positive thymocytes (i.e. L3T4+Ly‐2− or L3T4−Ly‐2+) during i.p. treatment with 15 mg/kg/day of CsA; (b) both populations of single‐positive thymocytes reappear within 2 weeks of termination of CsA and (c) irradiation and bone marrow reconstitution of these CsA‐treated mice results in reconstitution of normal numbers of L3T4+ and Ly‐2+ cells, but the L3T4+ T cells to not provide T helper function, as determined by interleukin 2 production and cytotoxic T lymphocytes generation. These findings indicate that CsA can affect thymic microenvironment and may be important as a model for investigating intrathymic T cell maturation. Our results may also have clinical implications for T lymphocyte development in transplant patients receiving CsA.
Alloreactive cytotoxic T cells induce DNA fragmentation in peritoneal macrophages: evidence for target cell killing by cytotoxic T cells in vivo
Authors: Liu Yang , Waring Paul , Müllbacher Arno
Published: March 2007

This report addresses the question whether cytotoxic T cells can cause target cell death in vivo by examining target cell DNA fragmentation. The results show that alloreactive cytotoxic T cells induced significant DNA fragmentation in peritoneal macrophages in vivo and that the DNA fragment was a multiple of 180 ± 30 bp. Furthermore, the effector cells which caused this characteristic DNA fragmentation were CD8+ T cells. These results demonstrate that cytotoxic T cells can cause target cell death in vivo.
Kinetic analysis of Ly‐6 gene induction in a T lymphoma by interferons and interleukin 1, and demonstration of Ly‐6 inducibility in diverse cell types
Authors: Leclair Kenneth P. , Bridgett Margot M. , Dumont Francis J. , Palfree Roger G. E. , Hämmerling Ulrich , Bothwell Alfred L. M.
Published: March 2007

The Ly‐6 locus contains multiple genes encoding cell surface proteins, two of which, when cross‐linked by antibodies, effect antigen‐independent activation of T lymphocytes. In this study, cDNA for Ly‐6‐encoded antigens have been used as probes to examine RNA from various tissues and transformed cell lines for constitutive levels of Ly‐6 RNA expression. Analyses of RNA prepared from several different tissues revealed a high level of expression of Ly‐6 RNA in kidney, spleen, heart and thymus, with a more moderate level of expression in liver, brain and lung tissue cells. A survey of various cell lines demonstrated the presence of Ly‐6 RNA in many, but not all T lymphocytic cell lines, in L cells, the Meth A fibrosarcoma, in the TCMK kidney cell line, and in the Neuro‐2a neuroblastoma. We also evaluated the expression of Ly‐6 RNA in cells after treatments with interferons (IFN) and interleukin 1 (IL 1). Treatment of lymphoid cells with IFN (α/β and γ), known to increase cell surface Ly‐6 antigen expression in normal T cells, was correlated with increases in Ly‐6 RNA levels. Increases in levels of RNA correlated with increases in levels of the Ly‐6A/E or Ly‐6C antigens. Several T lymphoid cell lines exhibiting Ly‐6 RNA inducibility by IFN were similarly inducible with IL 1. Kinetic experiments using one such line, (YAC‐1), showed that the induction of Ly‐6 RNA mediated by IFN‐α/β occurred rapidly (within 4 h), while the induction by IL 1 required relatively more time (∼︁ 8 h). Although the actions of IFN‐α/β were not blocked by cycloheximide, the presence of this protein synthesis inhibitor significantly attenuated the effects of IL 1 and IFN‐γ on Ly‐6 RNA transcription. Induction by IFN‐γ as well as IL 1 could be blocked completely by co‐culture with anti‐IFN‐γ, implicating IFN‐γ as a mediator of the induction by IL 1.
Lymph node macrophage heterogeneity: the phenotypic and functional characterization of two distinct populations of macrophages from rat lymph node
Authors: Chao David , MacPherson G. Gordon
Published: March 2007

The isolation and characterization of lymph node macrophages (MΦ) has shown a hitherto unknown heterogeneity. Two types of MΦ were distinguished by morphology, monoclonal antibody staining and functional assays. The type I MΦ failed to express surface Ia even when activated, a characteristic which has only previously been reported for splenic marginal zone MΦ despite studies suggesting an antigen presentation role for the MΦ, the failure to express surface Ia would seem to eliminate an interaction with T helper cells for the type I MΦ in the lymph node. In contrast, the type I MΦ, other characteristics of clustering with activated B cells in vitro, the colocalization of the type I MΦ and activated B cells in situ, the specific uptake of thymus‐independent type 2 antigens and the failure to undergo respiratory burst activity all suggest a MΦ‐B cell interaction, possibly of a trophic nature.

The defective microbicidal activity of the type I MΦ may have been compensated for by the type II MΦ, which expresses both strong respiratory burst activity and surface Ia expression when freshly isolated. However, unlike the inflammatory MΦ the activated phenotype of the type II MΦ did not appear to be interferon‐γ dependent because type II MΦ could also be isolated from nude rat lymph nodes.
Differential pattern of T cell recognition of the 65‐kDa mycobacterial antigen following immunization with the whole protein or peptides
Authors: Brett Sara J. , Lamb Jonathan R. , Cox Josephine H. , Rothbard Jonathan B. , Mehlert Angela , Ivanyi Juraj
Published: March 2007

The 65‐kDa stress protein from Mycobacterium bovis (Bacillus Calmette Guérin) elicited T cell proliferation and antibody responses in seven B10 congenic mouse strains with different H‐2 haplotypes. To analyze T cell determinants on this antigen, seven peptides corresponding to six predicted T cell epitopes, and one defined B cell epitope were synthesized. Mice were either immunized with the whole antigen and the specificity of the response was ascertained in respect of the six peptides, or mice were immunized with seven of the peptides and tested for proliferative responses to the whole molecule. The results showed that three peptides carried epitopes to which mice responded following injection of the whole molecule and that immunization with two additional peptides could prime for in vitro stimulation with the native antigen. The latter result indicates the feasibility of generating T cell responses to “cryptic” epitopes on proteins by immunizing with peptides. The peptide‐specific T cell responses were distinctly influenced by the H‐2 haplotype of mouse strains. However, two peptides were recognized by several H‐2‐disparate mouse strains, and one peptide could be presented by both I‐A and I‐E molecules. Immunization with several peptides induced a cross‐reactive T cell proliferative response to the homologous GroEL protein isolated from E. coli. The amount of cross‐reactivity was influenced by the extent of sequence homology between mycobacterial and E. coli proteins and the major histocompatibility complex class II molecule used to present the peptide.
Suppressive effect of interferon‐γ on the BCL1 cell‐dependent interleukin 5 bioassay
Authors: Lohoff Michael , Sommer Frank , Röllinghoff Martin
Published: March 2007

Proliferation of BCL1 cells, generally used for the measurement of murine interleukin (IL)5, is shown to be strongly suppressed by interferon (IFN)‐γ. This effect can be abrogated by the addition of anti‐IFN‐γ antibodies. Thus, the IL5 activity of culture supernatants derived from T cells simultaneously producing IFN‐γ and IL 5, can only be seen with BCL1 cells in the presence of anti‐IFN‐γ antibodies. If anti‐IFN‐γ antibodies are omitted, T cells which produce both, IFN‐γ and IL5, may mistakenly be grouped into the TH1 subtype producing only IFN‐γ, but no IL5.
Expression of disulfide‐linked and non‐disulfide‐linked forms of the T cell receptor γ/δ heterodimer in human intestinal intraepithelial lymphocytes
Authors: Spencer Jo , Isaacson Peter G. , Diss Tim C. , Macdonald Thomas T.
Published: March 2007

Immunohistochemistry has been used to investigate disulfide‐ and non‐disulfide‐linked forms of the T cell receptor γ/δ heterodimer (TcR γ/δ) in blood and intestinal epithelium of normal human small intestine, intestine of patients with untreated coeliac disease (in whom T cells expressing TcR γ/δ are disproportionately raised), intestine of patients with tropical malabsorption, and in the human fetus. In blood from adult volunteers, 90% of T cells expressing TcR γ/δ use the disulfide‐linked form. In contrast in the epithelium in normal small intestine, coeliac disease and tropical malabsorption, most of the T cells expressing TcR γ/δ use the non‐disulfide‐linked form. This is especially prominent in untreated coeliac disease where the increase in TcR γ/δ T cells is mainly restricted to those using the non‐disulfide‐linked form. In human fetal small intestinal epithelium, however, only cells using the disulfide‐linked form are present. These variations in expression of different forms of TcR γ/δ in the gut epithelium in different conditions suggests that antigen, or some as yet undefined factor may determine the frequency of each subpopulation.
Decline in growth and antibody production of established hybridomas and transfectomas with addition of interleukin 6
Authors: Karavodin Linda M. , Glasebrook Andrew L. , Phelps Julia L.
Published: March 2007

Several sources of the lymphokine interleukin (IL) 6 (human recombinant IL6, supernatant from P388D1, a murine macrophage cell line and murine thymocyte‐conditioned media) were examined for enhancement of growth, immunoglobulin (Ig) secretion and seeding density requirements in murine hybridoma and transfectoma cell lines established in the absence of exogenously added IL 6. None of these preparations increased growth or Ig secretion in the six cell lines studied. In fact, inhibition of proliferation was seen with two of the three IL 6 preparations. Human recombinant IL6 showed no effect on minimal seeding density, but murine thymocyte‐conditioned media gave a tenfold decrease in minimal seeding density requirements in one of two hybridomas and one of two transfectomas studied. All preparations contained active IL6, as measured by proliferation of the IL6‐dependent cell line T1165.D10. These data show that IL6 alone does not increase growth or Ig secretion for these established cell lines. This finding is compared with previous reports describing increased growth in primary cultures of murine hybridomas and human myelomas with IL6.
A cytokine, lymphocyte blastogenesis inhibitory factor (LBIF), arrests mitogen‐stimulated T lymphocytes at early G1 phase with no influence on interleukin 2 production and interleukin 2 receptor light chain expression
Authors: Sugimura Kazuhisa , Ueda Yutaka , Takeda Koji , Fukuda Shigeharu , Tsukahara Kappei , Habu Yoshiki , Fujiwara Hiroshi , Azuma Ichiro
Published: March 2007

The function of a human cytokine, lymphocyte blastogenesis inhibitory factor (LBIF), was characterized. To this end, LBIF was purified from crude supernatant of U‐937 cells, a human macrophage‐like cell line, by using fast protein liquid chromatography (FPLC).

We demonstrated here that (a) the LBIF preparation completely inhibited phytohemagglutinin (PHA)‐stimulated T cell proliferation; (b) however, in PHA‐stimulated T lymphocytes LBIF inhibited neither interleukin (IL)2 production nor the expression of IL2 receptor (IL2R) light chain (CD25) which play a critical role for T cell proliferation; (c) LBIF arrested PHA‐stimulated T lymphocytes at the G1 phase of cell cycle and inhibited entry into S phase, thus inhibiting lymphocyte proliferation. Wright‐Giemsa's staining of the cells showed that PHA/LBIF‐stimulated cells were arrested in early G1. In agreement with this result, LBIF strongly inhibited PHA‐induced RNA synthesis. Further, LBIF inhibited the induction of the transferrin receptor which is normally expressed at the late G1 phase of the cell cycle. The inhibitory activity of LBIF was reversible. Thus, this study elucidated that LBIF arrests PHA‐stimulated T lymphocytes at a point between the stages of IL 2 production or IL2R light chain expression (in early G1) and transferrin receptor expression (in late G1). Taken together, these results suggest that there might be a control system of T cell proliferation distinct from the previously reported mechanisms, such as the inhibition of IL2 production or the inhibition of IL 2R light chain (CD25) expression, and that LBIF might be an important molecule in the regulation of normal lymphocyte proliferation.
The biochemical characterization of E2, a T cell surface molecule involved in rosettes
Authors: Aubrit Françoise , Gelin Catherine , Pham Danièle , Raynal Brigitte , Bernard Alain
Published: March 2007

We have previously identified a molecule on the T cell surface, which, in addition to CD2 is involved in the rosette phenomenon. This is a 32‐kDa single polypeptide chain which we have termed E2. The studies reported here show striking patterns on the glycosylation status of E2. It is a heavily sialylated and glycosylated molecule, the sugar moieties accounting for almost half of its relative molecular mass (Mr). It carries no N‐linked sugar residues, only O‐linked oligosaccharides. Despite heavy glycosylation, the molecule appears to behave homogeneously on gel electrophoresis, both in terms of Mr and pI. Neuraminidase treatment of E2 lowered its Mr to 28 kDa; this was further decreased to 18 kDa after removal of O‐linked sugar residues by treatment with O‐glycanase. An identical reduction in size was observed after treatment with trifluoromethane sulfonic acid, showing that the molecule carries no detectable N‐linked sugar residues. Moreover, endoglycosidase F and endoglycosidase H treatment of either the immunoprecipitates from 125I surface‐labeled thymocytes, or of a purified preparation of E2, did not reduce the Mr of E2, nor did tunicamycin treatment of T cells. Two‐dimensional gel electrophoresis revealed two discrete spots of acidic pI (4.4 and 4.6) that were still seen after neuraminidase treatment, though they had moderately shifted. Pulse‐chase experiments revealed a single 28‐kDa precursor form that could have been the unsialylated molecule. Finally, sequencing 14 amino acid residues of the N‐terminal side revealed no homology with known proteins. Since the sugar moieties of adhesion protein could play an important role, the results obtained in this study will prove valuable to our understanding of the role exerted by the E2 molecule.
Bispecific F(ab′)2 monomer prepared with anti‐CD3 and anti‐tumor monoclonal antibodies is most potent in induction of cytolysis of human T cells
Authors: Nitta Taizo , Yagita Hideo , Azuma Takachika , Sato Kiyoshi , Okumura Ko
Published: March 2007

Induction of cytolytic activity of peripheral blood mononuclear cells towards target cells was studied by preparing bispecific F(ab′)2 which was composed of two Fab′ fragments, one of which was derived from anti‐CD3 monoclonal antibody and the other from anti‐tumor monoclonal antibody. After reduction of the interchange disulfide bonds of these fragments by dithiothreitol, a thiol‐disulfide interchain reagent, 5,5′‐dithiobis‐2‐nitrobenzoic acid, was added to convert the free SH groups of one of the Fab′ fragments to mixed disulfide derivatives. These were then coupled with the other Fab′ fragment bearing free SH groups, producing a bispecific hybrid F(ab′)2 monomer of high yield. The bispecific F(ab′)2 monomers were able to render nonactivated peripheral blood mononuclear cells cytotoxic against natural killer‐resistant tumor cell lines at doses as low as 1 μg/ml. The polymeric forms of the F(ab′)2 fragments prepared by use of cross‐linking reagents such as N‐succinimidyl‐3‐(2‐pyridyldithiol)‐propionate (SPDP) or S‐acetylmercaptosuccinic acid anhydride (SAMSA) were less efficient for induction of cytolytic activity than the monomeric one. It may be feasible to use bispecific F(ab′)2 monomers in cancer immunotherapy because of the ease of preparation, as well as the efficiency in inducing cytolytic activity and their high tissue permeability due to their small size (100–110 kDa). In addition, redirected cytolytic activity towards peripheral blood mononuclear cells by reticuloendothelial system cells, resulting from linking these two types of cells through Fc‐Fc receptor interactions, does not occur.
Cytoplasmic CD3+ surface CD8+ lymphocytes develop as a thymus‐independent lineage in chick‐quail chimeras
Authors: Bucy R. Pat , Coltey Monique , Chen Chen Lo H. , Char David , Douarin Nicole M. Le , Cooper Max D.
Published: March 2007

We have analyzed the embryonic development of a population of lymphoid cells that express a CD3 antigenic determinant in the cytoplasm but not on the cell surface. Since these cells lack T cell receptor (TcR) molecules, we have provisionally named them TCRO cells. Their development, expansion and distribution was investigated following transplantation of splenic and bursal fragments from chicken embryos into quail embryos. Since quail cells are not recognized by our panel of monoclonal antibodies against chicken TcR1, TcR2, TcR3, CD3, CD4 and CD8 molecules, these antibodies provided reliable markers for donor chick lymphocytes in the tissues of the quail recipients. Transplanted spleen and bursa both generated CD3+ cells, the number of which increased as a function of age. Notably, approximately half of these CD3+ cells expressed surface CD8, but none acquired TcR1 (γ/δ), TcR2 (α/β) or TcR3 expression. Since TCRO cells normally appear first in the spleen of 8‐day chick embryos (E8), their generation in E6 splenic transplants indicated an extrathymic origin. The TCRO cells of chick splenic origin migrated to the spleen, bursa and thymus of the quail recipients. In six of seven chimeras acquiring CT3+ cells in the recipient thymus, these cells were restricted to the medulla and displayed the typical TCRO phenotype: CD3+CD8+TcR1‐TcR2‐TcR3‐. These intrathymic TCRO cells also lacked the CT1 thymocyte antigen. We conclude that the TCRO cells represent a thymus‐independent lineage of lymphoid cells that can migrate into a receptive thymus but rarely, if ever, differentiate into conventional T cells.
Fate and biological action of human recombinant interleukin 1β in the rat in vivo
Authors: Klapproth Jan , Castell José , Geiger Thomas , Andus Tilo , Heinrich Peter C.
Published: March 2007

The plasma half life of recombinant human interleukin 1β (rhIL 1β) was determined in rats by measuring the disappearance of the radioactivity of 125I‐labeled rhIL 1β from the circulation. The plasma clearance showed a biphasic behavior: an initial fast disappearance (half life of about 3 min) was followed by a second slower one (half life of about 4 h). Twenty minutes after a single‐dose injection of 125I‐labeled rhIL 1β most of the radioactivity was concentrated in kidneys, liver and intestine.

rhIL 1β induced the synthesis of α1‐acid glycoprotein (AGP), α1‐cysteine proteinase inhibitor (CPI) and β‐fibrinogen mRNA in liver. Half maximal stimulation was elicited by approximately 3000 U of rhIL 1β per animal. The mRNA changes for AGP and CPI were followed by corresponding protein increases in serum. Twenty hours after rhIL 1β injection, serum AGP rose from 0.7 to 2.5 mg/ml. CPI increased from 0.3 to 1.9 mg/ml 25 h after administration of rhIL 1β. Within 20 h after rhIL 1β injection, albumin serum concentration showed a strong decrease, preceded by a reduction in hepatic albumin mRNA levels. Neither changes in albumin synthesis nor degradation can explain this decrease suggesting that other mechanisms such as increased transvascular permeability are involved.
Inhibition by chlorpromazine of lymphokine‐specific mRNA expression in human thymocytes
Authors: Schleuning Michael J. , Duggan Anne , Reem Gabrielle H.
Published: March 2007

This study was designed to determine the effect of the phenothiazine chlorpromazine (CPZ) on the activation of human thymocytes. We provide evidence that CPZ inhibits the accumulation of mRNA specific for the lymphokines interleukin 2, interferon‐γ, tumor necrosis factor α and the proto‐oncogene c‐myc; by contrast, the accumulation of mRNA specific for the α chain of the interleukin 2 receptor and the subsequent early expression of Tac antigen on the cell surface is not inhibited by CPZ. The inhibition of the expression of lymphokine‐specific mRNA results in a decrease in interferon‐γ synthesis and in inhibition of thymocyte proliferation as determined by the incorporation of [3H]thymidine. In addition, we show that activation of protein kinase C (PKC) in human thymocytes by 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) causes the phosphorylation of a protein of a molecular mass of approximately 75 kDa. The function of this protein is as yet not defined, but it is possible that it plays a role in the transduction of the signals to the nucleus which in turn elicit the expression of the genes coding for c‐myc and for the lymphokines required for thymocyte activation. We also demonstrate that CPZ, like the immunosuppressant drug cyclosporin A does not inhibit the phosphorylation of the 75‐kDa protein which is induced by the activation of PKC by TPA and does not affect phosphoinositide breakdown, indicating that it exerts its effect at a site distal to the activation of PKC. These observations demonstrate that CPZ has an immunoregulatory function in addition to its psychotropic activity.