bluuebunny/crossref_metadata_2025 huggingface dataset:
Microheterogeneity in the recognition of a HLA‐DR2‐restricted T cell epitope from a meningococcal outer membrane protein
Authors: Wiertz Emmanuel , van Gaans‐van den Brink Jacqueline , Hoogerhout Peter , Poolman Jan
Published: March 2007

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA‐DR2‐restricted T cell clones recognizing the 49‐61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3+CD4+CD8−TcRct/β+, were selected by restimulation with the synthetic peptide OMP(49‐61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine‐specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single‐residue substitution analogues of the minimal epitope OMP(50‐58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49‐61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, He 51 and Phe 54 act as MHC‐anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since He 51 and Phe 54 can be substituted by conservative residues [as OMP(50‐58) and OMP(49‐61) analogues] and with Ala [as OMP(49‐61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different Vα and Vβ genes (which encompass the putative MHC‐binding regions first and second complementarity‐determining regions, CDR1 and CDR2) different modes of interaction with the HLA‐DR2 molecule indeed are likely to occur.
Signal joint of immunoglobulin Vλ1‐Jλ and novel joints of chimeric V pseudogenes on extrachromosomal circular DNA from chicken bursa
Authors: Kondo Tokukazu , Arakawa Hiroshi , Kitao Hiroyuki , Hirota Yoshikazu , Yamagishi Hideo
Published: March 2007

We isolated extrachromsomal circular DNA from the bursa of 18‐day chick embryos and cloned their BamHI fragments into a phage vector. We found examples of the signal joint fused by Vλ1‐Jλ, rearrangement and the sequences homologous to Vλ1 segments that showed: (1) clustered V pseudogene (ΨV) germ‐line segments containing new ΨV26, (2) a head‐to‐tail duplication of ΨV13‐ΨV12 region and (3) chimeric structures composed of 5′‐Vλ1 and 3′‐ΨV segments. Both intrachromosomal tandem duplications and extrachromosomal circles may be generated by unscheduled DNA synthesis and recombination. The chimeric structure of Vλ1 joined with upstream ΨV segments suggests the involvement of V gene replacement for somatic diversification of the immunoglobulin genes in addition to a mechanism of segmental gene conversion.
Reconstitution of C.B‐17 scid mice with BALB/c T cells initiates a T helper type‐1 response and renders them capable of healing Leishmania major infection
Authors: Varkila Kari , Chatelain René , Leal Ludmila M. C. C. , Coffman Robert L.
Published: March 2007

C.B‐17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)‐like response characterized by delayed‐type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon‐γ (IFN‐γ), but no interleukin‐4 (IL‐4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN‐γ during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti‐IFN‐γ antibody prior to infection and reconstitution prevented healing and permitted the development of a Th‐2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL‐4, but very little IFN‐γ, after antigen stimulation in vitro. As few as 104 transferred T cells led to a Th1‐like response, suggesting that the IFN‐γ is of host rather than donor origin. The transfer of very high numbers (7.5 x 107) of BALB/c spleen cells overcame the effects of the IFN‐y and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measureable effect upon the development of a healing response in reconstituted scid mice.
Position 71 in the α helix of the DRβ domain is predicted to influence peptide binding and plays a central role in allorecognition
Authors: Coppin Hélène L. , Carmichael Paul , Lombardi Giovanna , L'Faqihi Fatima E. , Salter Russell , Parham Peter , Lechler Robert I. , De Preval Claude
Published: March 2007

Despite all the structural and functional data that have been accumulated regarding major histocompatibility complex (MHC) class II molecules during recent years, the relative contribution of putative T cell receptor (TcR)‐contacting residues and peptide‐binding MHC polymorphisms to MHC‐restricted and allospecific T cell responses remains a point of contention. Some authors emphasize the importance of direct interaction between the allospecific TcR and polymorphic MHC residues whereas other emphasize the role of naturally processed MHC‐bound peptides.

We have previously described a new HLA‐DRB1 allele: DR BON (DRB1*0103). This gene differs from DRB1*0101 by six base pairs clustered in the third variable region of the second exon leading to three amino acid changes at positions 67, 70 and 71 of the β chain of the HLA‐DR molecule. To define the respective role of these residues in allorecognition, we have performed site‐directed mutagenesis on the DRB1*0103 allele to create six mutants which are intermediary between the DR BON and the DR1 alleles. These mutant cDNA were expressed in mouse fibroblasts and the transfectants with the highest expression of class II molecules were used as stimulators for a panel of ten anti‐DR BON and five anti‐DR1 alloreactive T cell clones. We demonstrate that the residue at the peptide‐binding position 71 is of paramount importance in the alloresponse of these clones. In addition some clones were sensitive to amino acid substitution at the TcR‐contacting position 70, while substitution at position 67 affects very few clones. The dominance of residue 71 was also observed with an influenza hemagglutinin‐specific HLA‐DR BON‐restricted T cell line.
Limiting dilution analysis of interleukin‐2‐producing T cells responsive to recall and alloantigens in human immunodeficiency virus‐infected and uninfected individuals
Authors: Schulick Richard D. , Clerici Mario , Dolan Matthew J. , Shearer Gene M.
Published: March 2007

Peripheral blood mononuclear cells (PBMC) from individuals who were seropositive for the human immunodeficiency virus type 1 (HIV), and most without symptoms (HIV+) were compared with PBMC from healthy HIV‐seronegative (HIV−) individuals for in vitro generated T helper cell (Th) responses. Th function in bulk culture and limiting dilution analysis was assessed by IL‐2 production following stimulation with influenza A virus (FLU) or irradiated allogeneic PBMC (ALLO). We observed that the frequencies of FLU‐ and ALLO‐stimulated Th cells were not appreciably different in the PBMC of those HIV‐ individuals, and that they were also not different in the PBMC of those HIV+ individuals who responded to both FLU and ALLO in bulk culture. However, there was a severe drop in the Th frequency to FLU in HIV+ individuals who were unresponsive to FLU but responsive to ALLO by bulk culture. The PBMC of HIV+ individuals who were unresponsive by bulk culture to both FLU and ALLO exhibited a drastic reduction in the Th frequencies for both stimuli. These results demonstrate a concordance between Th functional analysis performed by limiting dilution and bulk culture. The results also indicate that the early selective loss in Th function to recall antigens is not likely to be due simply to a difference in frequencies of FLU‐ and ALLO‐stimulated Th cells present prior to the onset of Th dysfunction.
Activation of CD44 induces ICAM‐1/LFA‐1‐independent, Ca2+, Mg2+, —independent adhesion pathway in lymphocyte‐endothelial cell interaction
Authors: Toyama‐Sorimachi Noriko , Miyake Kensuke , Miyasaka Masayuki
Published: March 2007

We have established an endothelial cell line KOP2.16 from pooled mouse lymph nodes. Resting lymphocytes avidly bound to KOP2.16 and migrated underneath the cytoplasm. The binding was partly mediated by VLA‐4 and VCAM‐1, but apparently independent of CD44 since anti‐CD44 antibody examined failed to inhibit the binding. However, pretreatment of lymphocytes with anti‐CD44 resulted in the rapid appearance of Ca2+‐, Mg2+‐independent, LFA‐1/ICAM‐1‐, CD2/LFA‐3,VLA‐4/VCAM‐l‐independent lymphocyte binding, indicating that a novel adhesion pathway was induced by the anti‐CD44 treatment. Interestingly, the elicited adhesion was observed only when anti‐CD44 that block hyaluronate recognition of CD44 were used for lymphocyte pretreatment. Neither hyaluronate itself nor non‐blocking anti‐CD44 up‐regulated the adhesion. Fab fragment of the blocking anti‐CD44 did not induce the up‐regulation unless cross‐linked with a second antibody, indicating that cross‐linking of surface CD44 is necessary for induction of a novel adhesion pathway. We propose that the agonistic anti‐CD44 antibodies induce a novel adhesion pathway by mimicking ligand binding to CD44 on the lymphocyte surface and that non‐hyaluronate ligand(s) is involved in regulation of adhesive function of CD44. Potential involvement of such a regulatory mechanism in lymphocyte homing is discussed.
Expression of human minor histocompatibility antigen on cultured kidney cells
Authors: Beck Yoshifumi , Sekimata Masayuki , Nakayama Setsuko , Müller Gerhard A. , Müller Claudia A. , Yamamoto Junji , Nagao Takeshi , Uchida Hisanori , Akiyama Nobuo , Kariyone Ai , Takiguchi Masafumi
Published: March 2007

Incompatibility of human minor histocompatibility (hmH) antigens can induce rejection of grafts in organ transplantation and graft‐versus‐host reactions in bone marrow transplantation. In spite of their importance in clinical transplantation, hmH antigens are not well studied. Previous studies have demonstrated the expression of hmH antigens on Tand B cells, hematopoietic progenitor cells and keratinocytes. We have for the first time demonstrated the expression of hmH antigens on cultured kidney cells using HLA‐B35‐restricted, hmH antigen‐specific cytotoxic T lymphocyte (CTL) clones, which were previously established from a patient who rejected two kidneys from HLA‐identical sisters. The CTL clones could not kill cultured kidney cells. Since cultured kidney cells expressed very low levels of HLA class I antigens it was thought that their failure to be killed by the CTL clones was due to lack of expression of HLA‐B35 antigens. After induction of class I antigens on cultured kidney cells by interferon‐y (IFN‐γ), the IFN‐γ‐treated cultured kidney cells were killed by the CTL clones. Furthermore, we isolated hmH antigens as peptides from cultured kidney cells after treatment with IFN‐γ. These results indicate that cultured kidney cells express hmH antigens when HLA class I antigen is induced by IFN‐γ and hmH antigens on cultured kidney cells are recognized by T cells as peptides presented by HLA‐B35 molecules.
Manipulation of the superantigen‐induced lymphokine response. Selective induction of interleukin‐10 or interferon‐γ synthesis in small resting CD4+ T cells
Authors: Cardell Susanna , Höidén Ingmarie , Möller Göran
Published: March 2007

The production of several lymphokines by freshly isolated CD4+ T cells has been analyzed at the single‐cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) were induced, but very low frequencies of CD4+ T cells produced IL‐4, IL‐5 or IL‐10 in response to SEB. Exogenously added IL‐4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN‐γ production was reduced, while a high fraction of cells contained IL‐10 and IL‐4 after activation in the presence of IL‐4. We further demonstrate that IL‐4 and IL‐10 or IFN‐y production was selectively induced in resting, high‐density CD4+ T cells during primary stimulation, by SEB + IL‐4 or SEB. Under conditions where both IL‐10 and IFN‐γ were produced, most cells contained only one of the two lymphokines.
Interferon‐γ arrests proliferation and causes apoptosis in stromal cell/interleukin‐7‐dependent normal murine pre‐B cell lines and clones in vitro, but does not induce differentiation to surface immunoglobulin‐positive B cells
Authors: Grawunder Ulf , Melchers Fritz , Rolink Antonius
Published: March 2007

Normal pre‐B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin‐7 (IL‐7). Their IgH loci are partly in germ‐line, partly in DHJH‐rearranged configuration, while their light chain loci are in germ‐line configuration. They express the pre‐B cell‐specific genes VpreB and λ5. Proliferation of these pre‐B cells is inhibited by interferon (IFN)‐γ, with half‐maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre‐B cells exposed to IFN‐γ die by apoptosis, as is evidenced by the disintegration of pre‐B cell DNA into oligonucleosomal multimers of 180‐200 bp. While the proliferation of pre‐B cells from Eμ‐bcl‐2 transgenic (tg) mice is inhibited by IFN‐γ, these cells do not die by apoptosis. IFN‐γ does not induce differentiation to more mature B lineage cells. In the absence of IL‐7 normal pre‐B cells differentiate to VHDHJH/VLJL‐rearranged, surface immunoglobulin‐positive B cells expressing the a chain of the IL‐2 receptor. They also down‐regulate the expression of VpreB and X.5, and lose the capacity to proliferate on stromal cells in the presence of IL‐7. In contrast, both normal and Eμ‐bcl‐2 tg pre‐B cells exposed to IFN‐γ in the presence of stromal cells and IL‐7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of VpreB and λ5, do not express the α chain of the IL‐2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL‐7, once IFN‐γ is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B‐ALL) with IFN‐γ is discussed.
Human peripheral blood γδ T cells are uniformly sensitive to destruction by the lysosomotropic agents leucine methyl ester and leucyl leucine methyl ester
Authors: Pechhold Klaus , Kabelitz Dieter
Published: March 2007

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu‐OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3− CD16+ natural killer (NK) cells and a fraction of T cell receptor (TcR)αβ+ CD8+ T cells. We report that freshly isolated peripheral blood γδ T cells are highly sensitive to Leu‐OMe treatment as well. After incubation of PBMC with 5 mM Leu‐OMe or incubation of purified T cells with 50 μ.M leucyl leucine methyl ester (Leu‐Leu‐OMe) and subsequent overnight culture, CD3−CD16+ NK cells and γδ T cells were no longer detectable by immunofluorescence analysis. The two major γδ T cell subsets Vγ9+Vδ2+ and Vγ9‐Vδ1+ were equally susceptible to Leu‐OMe and Leu‐Leu‐OMe treatment. The elimination of Vγ9+ T cells by Leu‐OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of Vγ9+ T cells. In contrast, no activation of γδ T cells was elicited in Leu‐OMe‐treated responder T cells stimulated with killed mycobacteria.
Peripheral clonal deletion of superantigen‐reactive T cells is enhanced by cortisone
Authors: Lussow Alexander R. , Crompton Tessa , Karapetian Ochine , MacDonald H. Robson
Published: March 2007

The Tcell receptor (TcR) Vβ‐specific expansion, deletion and induction of nonresponsiveness among murine T cells responding to superantigens in the periphery has been well characterized. Here we demonstrate that clonal deletion of staphylococcal enterotoxin (SE) B‐reactive Vβ 8.2+ cells can be significantly increased when mice are injected with hydrocortisone (HC) following superantigen stimulation in vivo. The induced sensitivity to HC persists for at least 30 days after SEB injection, making it unlikely that proliferating cells were uniquely responsible for the enhanced deletion. Superantigen‐induced HC sensitivity was a general phenomenon and could also be observed among Vβ 11+ cells after the injection of SEA. Experiments conducted on thymectomized mice indicated that HC‐sensitive, SEB‐responsive cells could not be accounted for by rapidly produced, immature lymphocytes recently exported from the thymus. Further, Vβ 8.1+ peripheral lymphocytes from TcR transgenic mice expressing the Mls‐1a superantigen were sensitive to HC. These results imply that the majority of cells remaining after superantigen‐induced clonal expansion and deletion in vivo have indeed reacted with the superantigen. Implications for differential superantigen recognition by Tcells expressing the same TcR Vβ domain, perhaps due to a significant Vα contribution to the interaction in vivo, are discussed.
Human immunodeficiency virus gp120 and derived peptides activate protein tyrosine kinase p56Ick in human CD4 T lymphocytes
Authors: Hivroz Claire , Mazerolles Fabienne , Soula Mahdhia , Fagard Rémi , Graton Sophie , Meloche Sylvain , Sekaly Rafick P. , Fischer Alain
Published: March 2007

Human immunodeficiency virus binds to CD4 T lymphocytes by interaction between its envelope glycoprotein gpl20 and the CD4 molecule. The latter is non‐covalently associated with a src‐related tyrosine kinase, p56lck. CD4 cross‐linking increases the activity of p56lck, leading to phosphorylation of several cellular substrates. We report here that gpl60/120 increases both the autophos‐phorylation of p56lck and its enzymatic activity (reflected by phosphorylation of an exogeneous substrate) in normal T cells and the HUT78 CD4+ T cell line. This effect was detectable 5 min after activation and persisted for 40 min in normal T cells. It did not require gpl20 cross‐linking and was associated with phosphorylation of tyrosine residue on several proteins, as shown by phosphotyrosine Western blot analysis. The pattern of proteins phosphorylated on tyrosine residues in response to gpl20 activation was distinct from that induced by anti‐CD4 antibodies. p56lck activation required its association with CD4, since p56lck activity was not modified in HUT78 T cell lines expressing a truncated or mutated form of CD4 unable to associate with p56lck. Peptides mimicking residues 418 to 434 and 449 to 464 of HIV‐1 Bru gpl20, regions known to participate in gpl20 binding to CD4, also increased p56lck activity and triggered phosphorylation of similar substrates. Taken together, these results show that gpl60/120 and derived peptides can transiently increase p56lck activity without the need for CD4 cross‐linking. This activation led to a specific pattern of tyrosine phosphorylation on cellular proteins that may be of significance in the biological effects of the gpl20/CD4 interaction, e.g. syncytium formation and inhibition of T cell activation.
Peptide‐induced memory (IgG) response, cross‐reactive with native proteins, requires covalent linkage of a specific B cell epitope with a T cell epitope
Authors: Zegers Netty D. , van Holten Conny , Claassen Eric , Boersma Wim J. A.
Published: March 2007

In order to raise antibodies synthetic peptides are often coupled to a carrier protein to provide the necessary T cell determinants. Alternatively, a short synthetic determinant with a distinct sequence motif which can be presented by major histocompatibility complex (MHC) class II toT cells, can be linked directly to a B cell epitope. Recently, it has been suggested that covalent linkage between a class II‐presentable T helper peptide and a B cell epitope is not required to induce antibodies against a B cell determinant (Sarobe et al., Eur. J. Immunol. 1991. 21: 1555). Therefore, we investigated the ability of an H‐2d‐restricted T cell determinant (AA 111–120 FERFEIFPKEK) from the influenza virus hemagglutinin, to support B cell responses to different proven B cell determinant peptides, derived from human α1‐antitrypsin. Antibodies against B cell epitopes crossreactive with native α1‐antitrypsin could be raised only when these B epitope peptides were covalently coupled to the T cell determinant through a peptide bond. No antibodies were raised against the B cell epitope when the free peptides (Tand B cell epitopes) were just mixed or when the T cell epitope was conjugated via m‐maleimidobenzoyl succinimide ester or bis‐maleimidohexane to the B cell determinant. Antibodies against the T cell determinant were raised in all cases, regardless of the mode of presentation: just mixed with or covalently coupled to the B cell determinant. The results indicate that a covalent bond between T cell and B cell determinants in general is needed to induce anti B cell determinant antibodies cross‐reactive with the native protein.
Evaluation of the C‐terminal C5a effector site with short synthetic C5a analog peptides
Authors: Köhl Jörg , Lübbers Bettina , Klos Andreas , Bautsch Wilfried , Casaretto Monika
Published: March 2007

Biological activities have been determined for a series of 18 peptides based on the C‐terminal sequence of human or rat C5a. Lysosomal enzyme release was tested in two cell types, the promyelotic leukemia cell line U937 and polymorphonuclear leukocytes. In addition, an ATP‐release assay with guinea pig platelets was performed. It was demonstrated that the C‐terminal octapeptide 67‐74 of human C5a represents the minimal sequence required to induce a measurable biological signal in all assays. Extending this peptide to a length of 21 amino acids produced at best only a slight enhancement of potency. Amino acid replacements with either tryptophanyl or phenylalanyl residues in positions between 65–69 either increased potency (at position 67), or abrogated potency (at position 66) in the two lysosomal enzyme assays. N‐terminal acylation with the fluorenylmethoxy‐carbonyl‐aminohexanoyl group slightly enhanced C5a potency. In desensitization experiments with guinea pig platelets all peptides with a C5a activity were able to desensitize not only the C5a but also the C3a responses.
Monoclonal antibodies to the integrin α−4 subunit inhibit the murine contact hypersensitivity response
Authors: Chisholm Patricia L. , Williams Cynthia A. , Lobb Roy R.
Published: March 2007

Lymphocyte‐endothelial cell recognition is an active multistep process central to the pathophysiology of inflammation. In vitro models of lymphocyte adhesion predict that the β1 integrin very late antigen−4 (VLA−4), an activation‐dependent adhesion receptor, can mediate the firm sustained attachment required for the extravasation of memory lymphocytes. We have used murine contact hypersensitivity as an in vivo model in which to evaluate the role of α−4 integrins in an evolving inflammatory response. We demonstrate that the intravenous administration of 75μg of the anti‐α−4 specific monoclonal antibodies Rl‐2 or PS/2 4–6 h prior to challenge significantly inhibits the efferent response of 2,4 dinitrofluorobenzene, or oxazalone‐sensitized mice. The disease‐modifying effect of anti‐α4 treatment was evident as a 50–60% reduction in the ear swelling response. By histological analysis treated animals scored lower for edema, number of epidermal lesions and degree of leukocyte infiltration. Antibody‐treated animals have elevated numbers of circulating mononuclear leukocytes present in the same relative ratio as untreated control animals, suggesting that the inhibitory effect was not due to antibody‐dependent cellular depletion of effector lymphocytes. These data are consistent with a central role for VLA‐4 in the pathophysiologic process of inflammation.
Protection against autoimmune disease by bacterial agents. II. PPD and pertussis toxin as proteins active in protecting mice against experimental autoimmune encephalomyelitis
Authors: Ben‐Nun Avraham , Yossefi Sara , Lehmann Dan
Published: March 2007

Bordetella pertussis and Mycobacterium tuberculosis, routinely used to promote the development of autoimmune disease, were recently reported to also be effective in inducing protection against an autoimmune disease. Thus, we previously demonstrated that SJL/J and (SJL/J x BALB/c)F1 mice that are genetically susceptible to experimental autoimmune encephalomyelitis (EAE) become highly refractory to the induction of the disease following their exposure to B. pertussis and M. tuberculosis. In the present study, the pertussis toxin (PT) from B. pertussis and the purified protein derivative (PPD) of M. tuberculosis, were found to be sufficient to fully protect against EAE and thus may be the major bacterial components responsible for conferring protection. The 65‐kDa heat‐shock protein played only a marginal role in the protection against EAE induced by these bacteria. Both PT and PPD were protective when given before, but not after, the encephalitogenic challenge, and minute amounts (5–50 ng) emulsified in oil were sufficient to confer long‐lasting resistance to EAE. The effect of PT or PPD on EAE differed from that of mitogens or bacterial superantigens, suggesting that their protection ability was not attributable merely to mitogenic or superantigenic properties.

The mechanism of protection is not yet clear. Preliminary studies revealed a complex mechanism of protection whereby PPD and PT may operate differently. Thus, only PPD‐induced, but not PT‐induced, protection was transferrable by CD4+ T lymphocytes bearing an αγ Tcell antigen receptor. Neither PT nor PPD had a protective effect on EAE mediated by preformed pathogenic T lymphocytes and it is most likely that they exert their protection by affecting the development of such T lymphocytes. How bacteria such as B. pertussis and M. tuberculosis can either enhance the development of an autoimmune disease or protect against the disease is not yet clear. However, identifying PT and PPD as the bacterial components active in protection may allow a better understanding of the modulatory effects of bacteria and point to the potential use of such bacterial products in immunomodulation of autoimmune diseases.
APO‐1 mediated apoptosis or proliferation in human chronic B lymphocytic leukemia: Correlation with bcl‐2 oncogene expression
Authors: Mapara Markus Y. , Bargou Ralf , Zugck Christian , Döhner Hartmut , Ustaoglu Ferman , Jonker Richard R. , Krammer Peter H. , Dörken Bernd
Published: March 2007

We studied induction of apoptosis in several human malignant B cells from chronic lymphocytic leukemias (BCLL) by triggering the APO‐1 antigen. The APO‐1 antigen was found to be expressed on the surface of malignant B cells. In BCLL cells from most patients, APO‐1 antigen expression increased following in vitro activation by Staphylococcus aureus Cowan I (SAC) or interleukin‐2 (IL‐2). In certain cases of BCLL co‐stimulation with SAC plus IL‐2 resulted in a synergistic up‐regulation of the APO‐1 antigen on the cell surface and prepared BCLL cells for monoclonal antibody anti‐APO‐1 mediated apoptosis. Interestingly, bcl‐2 mRNA expression decreased upon stimulation with SAC plus IL‐2, whereas SAC or IL‐2 alone did not affect the level of bcl‐2 expression. Thus, in these BCLL cells induction of anti‐APO‐1‐mediated apoptosis appeared to be correlated with bcl‐2 mRNA down‐regulation. One informative BCLL, however, with a similar pattern of APO‐1‐antigen expression, did not show SAC plus IL‐2‐dependent bcl‐2 down‐regulation. Surprisingly, these cells proliferated in response to anti‐APO‐1 only when cells were co‐stimulated with SAC plus IL‐2. Our data suggest that down‐regulation of bcl‐2 prepares BCLL cells for induction of APO‐1‐mediated apoptosis. In addition they demonstrate that triggering of the APO‐1 antigen may also lead to the induction of proliferation in special cases of BCLL.
Role of cAMP in regulating cytotoxic T lymphocyte adhesion and motility
Authors: Valitutti Salvatore , Dessing Mark , Lanzavecchia Antonio
Published: March 2007

We investigated the functional role of the cAMP pathway in human cytotoxic T lymphocyte (CTL)‐target interaction. Pharmacological increase of intracellular cAMP concentration ([camp]i) inhibits killing, especially at low effectorto‐target ratios, suggesting an inhibitory effect on CTL recycling. We show that this inhibitory effect is primarily at the level of conjugate formation. Pharmacological increase in [cAMP]i, as well as treatment with cytochalasin D, results in a “rounding up” of the CTL and inhibition of the dramatic changes in shape that occur when a CTL forms a conjugate, even with an irrelevant target. In addition, pharmacological increase in [camp]i affects the cytoskeleton of the CTL since it induces a decrease of filamentous actin, as detected by flow cytometry on phalloidin‐stained CTL, and a stabilization of microtubules, as detected by increased resistance to the disrupting action of nocodazole.

In mature CTL (but not in their immature precursors), T cell receptor triggering by specific targets results in a measurable increase in cAMP levels and strongly synergizes with adenylyl cyclase activators such as prostaglandin E2, cholera toxin and forskolin. We suggest that T cell receptor triggering may induce accumulation of cAMP that interferes with cytoskeleton function and, thus, terminates CTL secretion and adhesion. These effects of cAMP are rapidly reversible and may regulate CTL recycling.
Peripheral selection of antigen receptor junctional features in a major human γδ subset
Authors: Davodeau François , Peyrat Marie Alix , Hallet Marie Martine , Houde Isabelle , And Henri Vie , Bonneville Marc
Published: March 2007

Recent studies demonstrating the existence of murine γδ T cell subsets with structurally identical T cell receptors (TcR) suggest that unlike αβ T cells, some γδ T cells are specialized in the recognition of a limited number of monomorphic antigens. However, this question still remains open in humans, since the TcR structural diversity of their peripheral γδ T cells was shown to be extensive. Here we have analyzed in detail the TcR chain genes expressed by human Vγ9+Vδ2+ peripheral blood lymphocytes (PBL), a major peripheral γδ T cell subset in adults and present evidence for an antigen‐driven peripheral selection of both TcR γ and δ junctional motifs among these cells. First, it is shown that the proportion of Vγ9+Vδ2+ cells expressing the V9JPC1 γ chain is much higher among PBL than among thymus‐derived clones, indicating that preferential use of this Jγ segment is not due to pairing or combinatorial constraints. Second, analysis of V9JPC1 γ transcripts derived from Vγ9+Vδ2+ PBL clones revealed a high prevalence of a unique V9JP γ sequence with limited “N” nucleotide additions and VJ trimming, which could not be accounted for by enzymatic or antigen‐independent structural limitations. Third, the TcR b chain expressed by most Vγ9+Vδ2+ PBL clones, though diverse in amino acid composition and length, carried a highly distinctive junctional motif, found at a much lower frequency among V2DJδ sequences derived from Vγ9−Vδ2+ PBL or Vγ9+Vδ2+ thymocytes. Together, these results which demonstrate shared γ and δ junctional features by cells using unique Vγ and Vδ genes, suggest that in vivo selection of Vγ9+Vδ2+ lymphocytes is mediated by a highly restricted number of nominal ligands.
Nucleotidic sequence analysis of the variable domains of four human monoclonal IgM with an antibody activity to myelin‐associated glycoprotein
Authors: Mariette Xavier , Tsapis Andréas , Brouet Jean‐Claude
Published: March 2007

We determined the nucleotide sequence of the VL and VH regions of four human monoclonal IgM directed to myelin‐associated glycoprotein (MAG) and a nerve glycolipid, the sulfated glucuronic paragloboside (SGPG). Clonal lymphoblastoid cell lines (three cases) and an heterohybridoma (one case) secreting anti‐MAG IgM were derived from patients' blood B cells.Vx genes derived from the single germinal VxIV (two cases), the VxId and the VxIIIa Humkv328h5 genes. Vh genes derived from the VhIII 9.1 germinal gene (or a closely related gene) in two cases, whereas two others possibly represent new members of the VhIII or VhI variability subgroups. There was no obvious restriction in the use of Jx Jh and Dh segments. Somatic mutations were predominantly found in the CDR3 of the VxIV genes with an overall ratio of replacement over silent mutations of 7/0. The sequence of two VhIII genes exhibited five replacement mutations in CDR in comparison to that of the germ‐line 9.1 gene.

Although some Vgenes are likely to be overrepresented among anti‐MAG IgM, the diversity of the immune repertoire for MAG and SGPG explains the lack of easily detectable public idiotopes among these IgM. This last finding, as well as a high ratio of replacement versus silent nucleotide mutations in the CDR of VL and probably VH genes, suggest that the pathogenesis of these monoclonal antibodies (and of the associated lymphoplasmocytic disorder) differs from that of other previously characterized monoclonal autoantibodies such as rheumatoid factors and cold agglutinins.
The major histocompatibility complex‐encoded proteasome component LMP7: alternative first exons and post‐translational processing
Authors: Glynne Richard , Kerr Lesley‐Anne , Mockridge Ian , Beck Stephan , Kelly Adrian , Trowsdale John
Published: March 2007

The LMP7 gene maps to the major histocompatibility complex class II region. The derived protein sequence shares homology with N‐terminal amino acid sequence from proteasome subunits (Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L.‐A. and Trowsdale, J., Nature 1991. 353: 357) and it has been suggested that LMP7 is involved in the degradation of endogenous antigens prior to their presentation through class I (Robertson, M., Nature 1991. 353: 300). We have isolated a second LMP7 transcript which has a different first exon to the published sequence. Both transcripts were expressed in cell lines from a number of tissues and both responded to inferferon‐γ. An anti‐LMP7 antiserum precipitated proteins similar in their migration on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to those precipitated by an anti‐proteasome serum. Western blot analysis of anti‐proteasome precipitates demonstrated that the LMP7 protein is incorporated into the proteasome but has a molecular mass of 23 kDa, 7 kDa smaller than expected from the derived protein sequence of either of the cDNA. A pulse‐chase experiment indicated that post‐translational cleavage of the LMP7 N terminus precedes the formation of the 23‐kDa proteasome subunit. To our knowledge, LMP7 provides the first biochemical evidence for such processing of proteasome components.
Identification of two distinct CD5‐ B cell subsets from human tonsils with different responses to CD40 monoclonal antibody
Authors: Dono Mariella , Zupo Simona , Masante Raffaella , Taborellin Giuseppe , Chiorazzp Nicholas , Ferrarini Manlio
Published: March 2007

This study investigated the response of different CD5− B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)‐4 or rabbit anti‐human μ chain antibody (a‐μ‐Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5+ B cells and subsequently fractionated on Percoll density gradients.

While resting CD5+ B cells proliferated and produced IgM molecules in response to a‐μ‐Ab, IL‐4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL‐2, resting CD5− B cells, which were co‐purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5+ B cells had the typical phenotype of mantle zone B cells (IgM+ IgD+ CD39+ CD38− CD10− CDw75dim), whereas resting CD5− B cells were CD38− CD39− CD 10− CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38+ CD10+ CD39− CDw75bright, IgG+) responded to CD40 mAb and IL‐4 and also to SAC plus IL‐2 further underlined the differences to resting CD5− B cells. However, some of the data collected suggest possible relationships between CD5− B cells and germinal center cells.

The CD5− B cells isolated from the 50 % Percoll fraction proliferated in response to a‐μ‐Ab, CD40 mAb and IL‐4 as well as to SAC and IL‐2. These cells had the same mantle zone B cell phenotype as the CD5+ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5+ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5− B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5− B cells did not express CD5.

The CD5− B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro.

The present study underlines the phenotypic and functional heterogeneity of CD5− B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.
CD2: a functional adhesion molecule on murine B cells, involved in interleukin‐4‐induced aggregation
Authors: Elenström‐Magnusson Carina , Altevogt Peter , Severinson Eva
Published: March 2007

The murine equivalent to CD2, previously known as a T cell marker, is expressed on mouse B cells. The monoclonal anti‐CD2 antibody 12‐15 was found to induce B cell homotypic adhesion. When treated with F(ab')2 fragments of 12‐15, purified, resting B cells aggregate within 2 h of incubation and the response is optimal after 20 h. Anti‐CD2‐induced aggregation is a dose‐related active process, dependent on temperature, metabolic energy and divalent cations. Aggregation is inhibited by two different Fab monomers of anti‐CD2, implying that it is the CD2 molecule itself that functions as an adhesion molecule. We also report that interleukin 4‐induced B cell homotypic adhesion involves CD2‐mediated cell binding, since the antibodies specific for mouse CD2, inhibited interleukin‐4‐induced cell aggregation.
Differential signaling through the Ig‐α and Ig‐β components of the B cell antigen receptor
Authors: Kim Kwang‐Myong , Alber Gottfried , Weiser Peter , Reth Michael
Published: March 2007

The B cell antigen receptor is a complex containing the antigen‐binding immunoglobulin molecules and the Ig‐α/Ig‐β heterodimer which presumably connects the B cell antigen receptor to intracellular signaling components. To analyze the functional properties of the cytoplasmic parts of the B cell antigen receptor, we used the K46 B lymphoma line (IgG2a, χ) to express chimeric molecules composed of the extracellular and transmembrane part of the CD8α molecule and the cytoplasmic sequence of either the Ig‐α (CD8α/Ig‐α), the Ig‐β (CD8α/Ig‐β) protein or the membrane‐bound γ2a heavy chain (CD8α/γ2a). From these three types of chimeric molecules only CD8α/Ig‐α and CD8α/Ig‐β, but not CD8α/γ2a, could transduce signals, thus providing the first evidence that the cytoplasmic tail of Ig‐α and Ig‐β have a signaling capacity. After cross‐linking with anti‐CD8α antibodies, both molecules induced a similar increase in intracellular free calcium ion and in MAP kinase phosphorylation. Protein tyrosine kinases, however, were strongly activated via the CD8α/Ig‐α and only marginally via the CD8α/Ig‐β molecule. This suggests that the Ig‐α and Ig‐β proteins have distinct roles during signal transduction through the B cell antigen receptor.
Human peripheral blood eosinophils produce and release interleukin‐8 on stimulation with calcium ionophore
Authors: Braun Ruedi K. , Franchini Marco , Erard Francois , Rihs Silvia , De Vries I. Jolanda M. , Blaser Kurt , Hansel Trevor T. , Walker Christoph
Published: March 2007

We present evidence that human blood eosinophils produce interleukin (IL)‐8 when stimulated with calcium ionophore. Following in vitro culture of 99 % pure eosinophils with calcium ionophore, released IL‐8 was detectable by enzymelinked immunosorbent assay in supernatants. Eosinophil IL‐8 production was considerably greater than that of IL‐3 or granulocyte macrophage colony‐stimulating factor. Furthermore, eosinophil production of IL‐8 in the presence of calcium ionophore could be inhibited with the immunomodulating agent cyclosporin A and the protein synthesis inhibitor cycloheximide. In addition, following stimulation of highly purified blood eosinophils with calcium ionophore, IL‐8 mRNA was detectable after polymerase chain reaction amplification. In comparison with other cells on stimulation with calcium ionophore, eosinophils produce about half as much IL‐8 as neutrophils but significantly more than purified T cells. In contrast to monocytes and neutrophils, IL‐8 production was not inducible with IL‐1 or tumor necrosis factor. Finally, following calcium ionophore stimulation blood eosinophils were shown to contain cytoplasmic IL‐8 by employing a monoclonal antibody against IL‐8 in conjunction with immuno‐histochemistry. These observations demonstrate that eosinophils are capable of IL‐8 production and release, which may contribute to defense against parasites and to the pathophysiology of allergic and asthmatic disease.
Human interleukin‐4 receptor signaling requires sequences contained within two cytoplasmic regions
Authors: Koettnitz Karl , Kalthoff Frank S.
Published: March 2007

The signaling pathway used by the murine or human interleukin‐4 receptor (WL‐4R) has not been elucidated so far. As an approach to mapping the cytoplasmic regions of the hIL‐4R that are essential for mediating the biological response upon IL‐4 binding we have cloned and expressed the wild‐type ML‐4R and several cytoplasmic deletion mutants in the murine IL‐3‐dependent pro‐B cell line BA/F3. Transfection of the wild‐type hIL‐4R conferred the ability on BA/F3 cells to proliferate in a dose‐dependent way when treated with human EL‐4. The analysis of six deletion mutants indicated that the signaling function of the hIL‐4R depends on sequences within two discontinuous regions that are located between amino acid He233 and Ser365 and Thr462 and Ala580 of the intracy toplasmic domain. The deletion of either of these regions totally abrogated IL‐4‐inducible growth. The relevance of our data is discussed in relation to the results obtaind from similar studies with other members of the hematopoietin receptor superfamily.
Peptides of 23 residues or greater are required to stimulate a high affinity class II‐restricted T cell response
Authors: Srinivasan Mallika , Domanico Susan Z. , Kaumaya Pravin T. P. , Pierce Susan K.
Published: March 2007

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen‐presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15‐residue peptide in an extended conformation is predicted to fit in an open peptide‐binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29‐residue cytochrome c peptide and a “promiscuous” peptide of tetanus toxoid, is more antigenic than the 23‐residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide‐binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.