bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro transformation by Epstein‐Barr virus induces a switch in growth factor and anti‐IgM responsiveness in a human leukemic B cell clone
Authors: Janssen Ottmar , Gillis Steven , Kabelitz Dieter
Published: March 2007

By in vitro transformation with Epstein‐Barr virus (EBV), we have previously established EBV lymphoblastoid cell lines (LCL) from a patient with leukemic centrocytic B cell lymphoma. EBV‐transformed LCL and EBV genome‐negative leukemic B cells showed identical chromosome aberrations and IgH gene rearrangements. In the present study we have analyzed the effect of exogenous cytokines [interleukin (IL) 1, 2, 3, 4, 6, tumor necrosis factor, lymphotoxin, transforming growth factor β, (TGF‐β)] and anti‐IgM antibodies on the in vitro proliferation of EBV− leukemic B cells and EBV‐converted LCL. In contrast to conventional chronic lymphocytic leukemia, B cells of the patient DUL spontaneously proliferated for up to two weeks in the absence of exogenous lymphokines. The spontaneous proliferative capacity of clonal DUL B cells was not modulated by IL 1, IL 3, IL 6, TNF or LT. In vitro growth of DUL B cells was increased, however, by exogenous recombinant (r)IL 2, and was abrogated by TGF‐β, rIL 4 and anti‐IgM. rIL 4 not only inhibited spontaneous B cell proliferation but also neutralized the enhancing effect of rIL 2. In contrast, growth of the EBV‐transformed DUL LCL was not affected by any of these factors. These data demonstrate that in vitro infection and transformation of a clonal B cell population by EBV induces a switch in responsiveness to rIL 4, TGF‐β and anti‐IgM. In addition, this report is the first to demonstrate an inhibitory effect of rIL 4 on a spontaneously proliferating human leukemic B cell clone.
Selective expression of Vδ6 genes by B2A2− CD4− CD− T cell receptor γ/δ thymocytes
Authors: Miescher Guido C. , Liao Nan Shih , Lees Rosemary K. , MacDonald H. Robson , Raulet David H.
Published: March 2007

Most CD4−CD8− adult murine thymocytes characterized by absence of the B2A2 (J11d) antigen express T cell receptors (TcR) α/β and utilize preferentially Vβ8.2 segments. To a much lesser extent, B2A2−CD4−CD8− thymocytes also express TcR γ/δ, as evidenced by biochemical and Northern blot analysis. We have now been able to exclude the possibility that these cells might co‐express both types of TcR: Vβ8 B2A2− CD4−CD8− thymocytes expressed no TcR δ mRNA whereas the corresponding Vβ8− subset transcribed full‐length TcR γ as well as δ mRNA. Furthermore, the TcR γ/δ expressing B2A2− thymocytes were found to use preferentially Vδ6 genes. Conversely, they did not express Vδ5 genes which are most frequently used by other TcR γ/δ‐bearing populations such as B2A2 CD4− CD8− thymocytes or CD4−CD8− peripheral lymph node cells. RNase protection experiments showed that two particular Vδ6 transcripts predominate in these γ/δ populations, the most prominent Vδ6 sequence being highly homologous if not identical to Vα7.1. Our observations extend previous information on overlapping Vα and Vδ gene repertoires, particularly in the cross‐hybridizing Vα7/Vδ6 gene family. Moreover, our data suggest that B2A2−CD4−CD8− thymocytes represent a developmentally unique subset in which both Vδ and Vβ segments are nonrandomly expressed.
Immigration of thoracic duct B lymphocytes into established germinal centers in the rat
Authors: Vonderheide Robert H. , Hunt Simon V.
Published: March 2007

Immigration of B lymphocytes into established germinal centers in the rat was studied by transferring genetically marked thoracic duct B cells to non‐irradiated congenic hosts at various times between 3 days before and 6 days after host immunization. Seven days after host immunization, the distribution of donor B cells to lymph node germinal centers (relative to their distribution to non‐germinal center lymph node areas) was measured by two‐color flow cytometry in which (a) donor and host B cells were distinguished by their Ig ϰ chain allotypes, and (b) germinal center B cells were distinguished by their lack of labeling with the monoclonal antibody HIS22. Thoracic duct B cells from long‐term antigen‐primed rats were found to immigrate into host germinal centers much better than B cells from unprimed donors. This effect was antigen specific: primed B cells only immigrated well into host germinal centers induced by the priming antigen. Although B cells localized in germinal centers most efficiently when injected before immunization, specifically primed donor B cells injected after immunization were still found to be at least as evenly distributed to germinal centers as to other lymph node areas, whereas unprimed B cells transferred after immunization localized poorly in host germinal centers. These findings are discussed in light of recent suggestions that memory B cell clones are maintained by continued antigenic stimulation within secondary lymphoid follicles.
The CD59 antigen is a structural homologue of murine Ly‐6 antigens but lacks interferon inducibility
Authors: Philbrick William M. , Palfree Roger G. E. , Maher Stephen E. , Bridgett Margot M. , Sirlin Sonia , Bothwell Alfred L. M.
Published: March 2007

A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K‐562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross‐hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly‐6E and Ly‐6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol‐glycan modification site for Ly‐6 molecules. Unlike Ly‐6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well‐characterized mouse Ly‐6 proteins.
Immunohistology of T cell differentiation in the thymus of H‐Y‐specific T cell receptor α/β transgenic mice
Authors: van Ewijk Willem , Kisielow Pawel , von Boehmer Harald
Published: March 2007

We examined the immunohistological aspects of the H‐Y specific T cell receptor (TcR) α/β transgene expression in the thymus of male and female transgenic (Tg) mice. Virtually all thymocytes expressed the β transgene in both the male and female thymus. Expression of accessory molecules (co‐receptors) in Tg mice deviated from control mice. In the male Tg thymus, CD8 expression was either low or absent on both cortical and medullary thymocytes. In contrast, in the thymus of female mice, CD8+ cells were found both in the cortex and in the medulla. The majority of medullary thymocytes was bright CD8+. This is in clear contrast to the CD8 distribution in control B6 mice, where only a few percent of medullary cells are CD8+. Similarily, the proportion of cells expressing CD4 antigens was reduced in the cortex and medulla of the thymus from male Tg mice, as compared to the thymus of female Tg mice and B6 control mice.

Comparative analysis of the stromal cell types of the thymic microenvironments in the three groups of mice revealed that the cortical thymic microenvironment of male Tg mice differed, compared to that of female Tg mice. In particular, the deep cortex showed a closely packed meshwork of epithelial reticular cells. Moreover, H‐2Db molecules (which are the restricting elements for the Tg TcR α/β) were abnormally expressed in the thymic cortex of male mice. The cortical microenvironment in female mice, on the other hand, appeared normal.

Together, the data indicate that TcR α/β transgene expression in male mice leads to an aberrant co‐receptor expression in both cortical and medullary lymphoid cells as well as an abnormal composition of the cortical microenvironment. Both phenomena may be the consequence of “negative selection” of developing H‐Y‐specific T cells, as it occurs only in the male Tg thymus. The absence of the H‐Y antigen, but presence of the restricting element H‐2Db in the thymic cortex of female mice, leads to accumulation of CD8+ in the medulla, a phenomenon interpreted as “positive selection”.
Reconstitution of H‐2 class I expression by gene transfection decreases susceptibility to natural killer cells of an EL4 class I loss variant
Authors: Sturmhöfel Knut , Hämmerling Günter J.
Published: March 2007

Several reports have suggested that an inverse correlation exists between major histocompatibility complex class I expression and the susceptibility to natural killer (NK)‐mediatedlysis. For example, the increased class I expression induced by interferon‐γ was always accompanied by an increased resistance to NK lysis. Likewise, class I loss variants were often more NK susceptible than their normal counterparts.

To investigate whether the inverse correlation between class I expression and NK susceptibility was fortuitous or whether the class I molecules were directly responsible for this effect we resorted to gene transfection studies. From the murine thymoma line EL4 an H‐2Db‐ and Kb‐negative variant S3 was selected. This variant was highly susceptible to NK lysis. S3 was found to have a defect in β2‐microglobulin gene expression. Therefore, restoration of Db and Kb expression could be achieved by transfection with the β2‐microglobulin gene. This resulted in a strong decrease in susceptibility to NK lysis to the level of the H‐2+ parental EL4. Transfection with class II genes had no effect. Blocking of the class I molecules on the H‐2+ cells with anti‐H‐2b F(ab')2 fragments increased the susceptibility to NK cells to the level of the H‐2− variant S3. These data demonstrate that the class I molecules on the targets are directly responsible for regulation of NK susceptibility but the mechanism is not clear. Possibly the class I molecules interfere with the unknown NK target structures.
Minor lymphocyte stimulating (Mls) gene products in mice influence their genetic resistance or susceptibility to induction of autoimmune encephalomyelitis
Authors: Ben‐Nun Avraham , Soffer David
Published: March 2007

The role of the major histocompatibility complex (MHC) gene products in the genetics of experimental autoimmune encephalomyelitis (EAE) is well established. Here we demonstrate how non‐MHC gene products, stimulatory to T cells specific to myelin basic protein (MBP), can affect the MHC control in determining genetic susceptibility or resistance to induction of EAE. I‐As‐restricted MBP‐specific T cells derived from SJL/J mice are shown to cross‐react with Mls‐2a gene products. The Mls‐2a gene product expressed by (SJL/J×BALB/c)F1 mice tolerize T cells recognizing I‐As/MBP and favor the development of I‐Es/d‐restricted MBP‐specific T cells mediating EAE in the (SJL/J×BALB/c)F1 mice. These I‐Es/d/MBP‐specific T cells, cross‐reactive with Mls‐1a, and the I‐As/MBP‐specific T cells, cross‐reactive with Mls‐2a gene products, are both eliminated by self tolerance mechanisms in the H‐2‐matched (SJL/J×DBA/2)F1 mice, expressing Mls‐1a2a gene products, and thereby confer genetic resistance to EAE on the (SJL/J×DBA/2)F1 mice bearing EAE‐permissive MHC alleles. These results reflect a developmental selection of a T cell repertoire to the self antigen MBP, imposed by self tolerance to self Mls gene products, which affect the genetic susceptibility to EAE. These studies also demonstrate that self tolerance to Mls gene products can strengthen the tolerance to organ‐specific self antigens such as MBP, which may not be expressed or which are absent in the thymus at the time of thymic selection.
Functional properties of CD19 B lymphocytes positively selected from buffy coats by immunomagnetic separation
Authors: Funderud Steinar , Erikstein Bjørn , Åsheim Hans Christian , Nustad Kjell , Stokke Trond , Blomhoff Heidi Kiil , Holte Harald , Smeland Erlend B.
Published: March 2007

Here we report that human B lymphocytes can be positively selected directly from buffy coats applying the anti‐CD19 antibody AB1 coupled to magnetic beads. This isolation protocol is highly efficient and the isolated cell population is of very high purity and viability. As judged by cell cycle analysis and various parameters for cell activation, the cells are still in a resting state after isolation. Furthermore, different functional assays have shown that the isolation procedure does not interfere with either activation or proliferation/differentiation of CD 19 selected cells as compared to negatively isolated cells. As a consequence of cross‐linking during the isolation process, the CD 19 antigen is temporarily down‐regulated as measured by AB1 binding. Despite this decreased expression, monoclonal antibodies to the CD19 antigen nevertheless inhibited anti‐μ plus B cell growth factor induced B cell activation as reported also for negatively isolated cells. Taken together, the presented data strongly suggest that B cells isolated through the CD 19 antigen can be used in critical functional assays.
Distinct γ/δ T cell receptors define two subsets of circulating porcine CD2−CD4−CD8− T lymphocytes*
Authors: Hirt Werner , Saalmüller Armin , Reddehase Matthias J.
Published: March 2007

The species swine provides the only example for CD2+ and CD2− subsets of lg−CD4−CD8− lymphocytes with the propensity for homing to lymphoid tissue (Saalmüller et al., Eur. J. Immunol. 1989. 19: 2011). That the CD2−CD4−CD8− lymphocytes are bare of marker molecules that typify T lymphocytes raised the question of whether or not this cell type is descended from the T lymphocyte differentiation lineage. It is documented that expression of a phylogenetically conserved external epitope of T cell receptor γ/δ subdivides porcine CD2− lymphocytes into an epitope 86D+ minor and an 86D− major subset. Expression of distinct forms of the T cell receptor γ/δ, disulfide‐bonded N‐glycosylated surface heterodimers of under reducing conditions 38/40 and 37/40 kDa, respectively, hallmarks the CD2−86D+ and CD2−86D− subsets both as T lymphocytes.
Phenotype of intraepithelial lymphocytes in euthymic and athymic mice: Implications for differentiation of cells bearing a CD3‐associated γδ T cell receptor
Authors: de Geus Bernard , van den Enden Margit , Coolen Co , Nagelkerken Lex , van der Heijden Philip , Rozing Jan
Published: March 2007

This study was carried out to determine the exact phenotype of intraepithelial lymphocytes (IEL) in euthymic and athymic nude mice. The phenotype of IEL in euthymic and athymic mice is mainly CD3+CD8+. However, based on Thy‐1‐and CD3‐associated receptor expression we can subdivide the CD3CD8 population into different subpopulations in euthymic and athymic mice. In euthymic and athymic mice several CD3CD8 populations can be defined. One population expressing Thy‐1 and the T cell receptor (TcR) αβ is absent in athymic mice. Two other CD3+CD8+ populations can be detected in euthymic and athymic mice. Based on Northern blot and flow cytometric analysis we have to conclude that these populations express the CD3‐associated TcR γδ. One of the TcR γδ‐expressing populations also expresses Thy‐1 at low surface density. This is in contrast to the CD3CD8 population expressing the TcR αβ, which expresses Thy‐1 at high surface density. There are also, however, especially in athymic nude mice, significant numbers of CD3−CD8+ cells present with the same localization as IEL. The function of these cells is yet unknown. Using a probe for the δ chain we have shown that IEL preferentially express 2‐kb mRNA, while nearly no δ chain 1.7‐kb mRNA is expressed by these cells. This is in contrast to δ mRNA in thymocytes. Equal quantities of the 1.7‐ and 2.0‐kb δ chain mRNA species were found in RNA isolated from thymocytes.

The results imply that CD3+CD8+ intestinal IEL expressing the CD3‐associated TcR γδ can differentiate in absence of the thymus and represent a thymusindependent lineage of cells bearing this receptor.
Low‐temperature inhibition of antigen processing and iron uptake from transferrin: Deficits in endosome functions at 18 °C
Authors: Harding Clifford V. , Unanue Emil R.
Published: March 2007

Antigen processing involves endocytosis, proteolysis and denaturation of antigens to generate peptides that bind to major histocompatibility complex class II molecules (Ia) in a complex recognized by CD4+ T cells. Ia and antigen are internalized and processed intracellularly, but the exact subcellular site of antigen degradation and formation of the Ia‐peptide complex remains unclear. The present studies utilized low‐temperature incubation in an attempt to functionally block certain steps in the processing of the antigen hen egg white lysozyme (HEL) by peritoneal exudate cells (PEC) and TA3 B lymphoma cells. Ia endocytosis and uptake of HEL by PEC persisted at 18 °C, albeit at somewhat slower rates, but delivery of ligands to lysosomes was blocked. Under these conditions HEL catabolism and antigen processing were effectively blocked, although enough HEL was internalized at 18 °C to provide effective presentation during a subsequent incubation at 37 °C. In TA3 cells transferrin endocytosis and recycling were notably slowed at 18 °C, and iron uptake from transferrin by TA3 cells was completely blocked, indicating that certain specifically endosomal functions were inhibited at 18 °C. Thus, intracellular steps in antigen processing were blocked at 18 °C, corresponding to deficits in endosomal processing and targeting. These results demonstrate that antigen endocytosis and certain temperature‐sensitive endosomal and lysosomal processes are essential for antigen processing.
Structural characterization of human monoclonal cold agglutinins: Evidence for a distinct primary sequence‐defined VH4 idiotype
Authors: Silverman Gregg J. , Carson Dennis A.
Published: March 2007

Cold agglutinins that bind the developmentally regulated I red cell determinant occur naturally among human monoclonal IgM proteins. These autoantibodies are known to use light chains that derive mainly from the minor kappa III (xIII) variable region subgroup. The xIII subgroup is also highly expressed in monoclonal rheumatoid factors. However, while most monoclonal rheumatoid factors use structurally homologous heavy chains that derive from the VH1 family, information regarding the structure of the cold agglutinin heavy chains remains fragmentary. We demonstrate here that the xIII cold agglutinin autoantibodies exclusively use heavy chains that derive from the VH4 family. Furthermore, these autoantibody heavy chains all express the same primary sequence‐defined idiotype, corresponding to the second hypervariable region. These data indicate that cold agglutinins use a remarkably homogeneous subset of heavy chain variable regions. Moreover, unique patterns of preferential VH and VL pairing clearly distinguish the anti‐I cold agglutinins from all other known monoreactive autoantibodies.
The role of CD44, CD45, CD45RO, CD46 and CD55 as potential anti‐adhesion molecules involved in the binding of human tonsillar T cells to phorbol 12‐myristate 13‐acetate‐differentiated U‐937 cells
Authors: King Philip D. , Batchelor Adrian H. , Lawlor Patrick , Katz David R.
Published: March 2007

In this study, we have shown that human tonsillar T cells adhere to phorbol 12‐myristate 13‐acetate(PMA)‐differentiated U‐937 cells. To examine the molecular mechanisms involved, the effect of a panel of monoclonal antibodies upon this adhesion was assessed in a quantitative binding assay. Antibodies against LFA‐1 and ICAM‐1 inhibited binding, directly implicated these molecules in T cell‐PMA‐induced U‐937 adhesion. Furthermore, the adhesion was magnesium but not calcium dependent. Of the remaining antibodies that were tested, none of those against CD2, LFA‐3, Mac‐1, p150,95, CD43, CD45RA or CD56 affected binding. However, antibodies against CD44, CD45, CD45RO, CD46 and CD55 enhanced binding suggesting an anti‐adhesive role for these molecules during U‐937‐T cell interaction.
Macrophage‐secreted prostaglandin E2 potentiates immune complex‐induced B cell unresponsiveness
Authors: Stein Sidney H. , Phipps Richard P.
Published: March 2007

Immune complexes (IC) are potent modulators of immune responses. In this report, we used an in vitro murine model system to investigate how two types of accessory cells pulsed with IC regulated B cell function. We demonstrate that IC‐pulsed macrophages (MΦ) induce hapten‐specific B cell unresponsiveness, whereas IC‐pulsed splenic lymphoid dendritic cells (LDC) and an LDC‐like tumor line caused an augmention of the antibody response. The mechanism by which IC‐pulsed MΦ diminished antibody production required two signals. The first was an antigen‐specific signal supplied by the IC, and the second a nonspecific co‐factor which was a product of MΦ cyclo‐oxygenase metabolism. Specifically, it was shown that prostaglandin E2 (PGE2) could function as this co‐factor. Interestingly, other prostanoids, such as PGF2α, did not function in this fashion. Purified fluorescein (FL+)‐specific B cells pulsed with IC exhibited a similar pattern of hapten‐specific unresponsiveness. Treatment of accessory cell‐free, FL+ B cells with PGE2 rendered them sensitive to subtolerogenic doses of soluble IC. Overall, our results suggest that one mechanism by which unresponsiveness can be induced involves both IC and PGE2, and that elevated levels of PGE2 sensitize B lymphocytes to antigen‐specific tolerogenic signals.
T cell receptor (TcR) β chain transgenic mice: Studies on allelic exclusion and on the TcR+ γ/δ population
Authors: Pircher Hanspeter , Ohashi Pamela , Miescher Guido , Lang Rosemarie , Zikopoulos Athanasios , Bürki Kurt , Mak Tak W. , Robson MacDonald H. , Hengartner Hans
Published: March 2007

To study allelic exclusion of TcR genes we analyzed two types (I and II) of TcR β transgenic mice. T cells derived from both types of mice contained similar amounts of transgenic RNA transcripts; however, surface expression of the transgenic β chain was drastically reduced in type II compared to type I. In type I transgenic mice, productive rearrangements and expression of endogenous TcR β genes were suppressed whereas on T cells of type II mice, both transgenic and endogenous TcR β chains were expressed on the surface of the same cell. These findings suggest that allelic exclusion of TcR genes in β transgenic mice depends on amount and/or onset of transgene expression during thymic development. Furthermore, TcR γ rearrangements and the population of TcR γ/δ‐bearing double‐negative CD4−CD8− thymocytes were reduced fivefold in type I transgenic animals. However, the Vγ usage and the γ/δ+ dendritic epidermal cell populations appeared normal. RNase protection analysis further revealed low levels of transgenic TcR β chain transcripts in TcR+ γ/δ CD4−CD8− thymocytes. These results suggest that the β transgene only quantitatively influences the γ/δ T cell compartment, and supports the independence of the γ/δ population.
Interleukin 7 is a growth factor for mature human T cells
Authors: Londei Marco , Verhoef Adrienne , Hawrylowicz Catherine , Groves Jill , de Berardinis Piergiuseppe , Feldmann Marc
Published: March 2007

We have studied the capacity of interleukin (IL) 7 to support the growth and expansion of human T cell clones of different phenotype and function. All the clones studied (CD4+CD8+, CD4−CD8− T cell receptor α/β or γ/δ) responded to IL 7. The proliferative response of all the T cell clones induced by IL 7 was routinely less than to IL 2, but comparable to the IL 4 response. IL 7 also induced the proliferation of resting, freshly prepared peripheral blood mononuclear cells (PBMC) or T cell‐enriched (E+) cells. The pattern of proliferation observed in the presence of IL 7 was similar, but lower in magnitude, to that induced by IL 2. In both these cells populations the response to lymphokines alone was always less than the response to lymphokines plus insolubilized anti‐CD3 monoclonal antibody. In contrast IL 4 produced a different pattern of responsiveness, as significant proliferation was observed only on PBMC costimulated with anti‐CD3. The possibility that IL 7 mediates its growth stimulation by the IL 2 pathway was excluded by the incapacity of anti‐IL 2 or anti‐Tac monoclonal antibody, in concentrations which blocked IL 2‐dependent proliferation, to inhibit IL 7‐dependent growth. We conclude that IL 7 is a major growth factor for human mature T cells, and its activity is not limited to lymphocyte progenitors.
Regulation of Ig gene expression in normal lymphocytes. I. The half‐life of secreted μ chain mRNA differs from that of membrane μ chain mRNA in resting and activated B cells
Authors: Berberich Ingolf , Schimpl Anneliese
Published: March 2007

Resting and activated B lymphocytes were used to study the stability of μ‐specific precursor and mature mRNA. Resting cells which predominantly process the μ precursor towards μm do so rather slowly as reflected in a precursor half‐life (T1/2) of 1‐2 h. The small amount of secreted μ chain mRNA is fairly stable (T1/2 ≈ 8 h) compared to membrane μ chain (T1/2 ≈ 4 h). After activation the precursor processing is very fast (T1/2 ≈ 10 min) and the stability of μ2, which now predominates, increases (T1/2 ≈ 16 h) while the half‐life of μm remains at about 4 h. The data indicate that normal B cells regulate μ‐specific mRNA stability differently from tumor cells of the B cell lineage.
Structural nature of the interaction between T lymphocyte surface molecule CD4 and the intracellular protein tyrosine kinase Ick
Authors: Vega Miguel A. , Kuo Mei‐Chang , Carrera Ana C. , Strominger Jack L.
Published: March 2007

The strong non‐covalent interactions between T lymphocyte surface CD4 or CD8 molecules and the intracellular membrane‐associated protein tyrosine kinase Ick are likely to mediate the role of CD4 and CD8 molecules in the immune response. The delineation of the structural nature of the CD4/Ick and CD8/Ick complexes is important for the understanding of the biochemical and functional significance of the interactions. Complementary charged regions in the C‐terminal intracyto‐plasmic portions of CD4 or CD8, and in the N‐terminal region of protein tyrosine kinase Ick were noted. Peptides spanning these regions, residues 417 to 429 of CD4 and 10 to 22 of Ick, were found to specifically dissociate these two molecules in CD4/Ick complexes. A structural model of the interaction that accounts for its high stability is proposed.
Identification of two promiscuous T cell epitopes from tetanus toxin
Authors: Ho Paul C. , Mutch David A. , Winkel Ken D. , Saul Allan J. , Jones Graham L. , Doran Trevor J. , Rzepczyk Christine M.
Published: March 2007

Tetanus toxoid‐specific T cell clones were isolated from a human donor. To determine the T cell epitopes recognized by the clones, 30 peptides representing amphipathic alpha helical regions of the tetanus toxin were screened for ability to induce proliferation of the clones. Two T epitopes were identified. These occurred within peptides 12 and 21, and had the amino acid sequences NSVDDALINSTKIYSYFPSV and PGINGKAIHLVNNESSE, respectively. An unusual feature was that both peptides could be presented to their respective T cell clones by antigen‐presenting cells of many HLA specificities. Further investigation of peptide 12 showed that the epitope was only seven amino acids in length and had a very hydrophobic sequence, namely YSYFPSV. The ability of the T cell epitope‐containing peptides 12 and 21 to interact with many different HLA alleles means they may potentially be very useful as “universal carrier molecules” in synthetic vaccines.
The peritoneal Ly‐1 (CD5) B cell repertoire is unique among murine B cell repertoires
Authors: Lalor Paul A. , Morahan Grant
Published: March 2007

Ly‐1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly‐1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain‐treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluoresence‐activated cell sorter. The peritoneal Ly‐1 B cell repertoire was found to be different from that of conventional B cells, with between 5‐ and 100‐fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly‐1 B cell anti‐haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen‐reactive antibodies within the Ly‐1 B repertoire are most likely explained by the presence of clones secreting low‐affinity or multireactive antibodies. The Ly‐1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly‐1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly‐1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly‐1 B cells in germ‐free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.
Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell‐mediated immunity
Authors: Lider Ofer , Mekori Yoseph A. , Miller Ted , Bar‐Tana Ruth , Vlodavsky Israel , Baharav Ehud , Cohen Irun R. , Naparstek Yaakov
Published: March 2007

Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell‐mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed‐type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 μg) or present in vitro (0.1 μg/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell‐mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.
Cytokine‐related syndrome following injection of anti‐CD3 monoclonal antibody: Further evidence for transient in vivo T cell activation
Authors: Ferran Christiane , Sheehan Kathleen , Dy Michel , Schreiber Robert , Merite Sylvie , Landais Paul , Noel Laure‐Hélène , Grau Georges , Bluestone Jeffrey , Bach Jean‐François , Chatenoud Lucienne
Published: March 2007

In vivo injection of the hamster anti‐murine CD3 monoclonal antibody 145 2C11 into BALB/c mice induces a massive systemic release of several cytokines. Very high circulating levels of tumor necrosis factor are detected both by enzyme‐linked immunosorbent assay and L‐929 bioassay 90 min following a single injection of 10 μg/mouse 145 2C11. Peak circulating levels of exclusively T cell‐derived products such as interferon‐γ, interleukin 2 and interleukin 3 are also detected 90 min to 8 h post‐injection. Importantly, this cytokine release is transient since none of these cytokines are still present 12 to 24 h post‐injection.

In parallel to cytokine release, 145 2C11‐treated mice (10 μg/mouse) exhibit somnolence, hypomotility (quantified by actimetry), hypothermia, diarrhea and piloerection. At this dosage, the physical reaction is not lethal and reverses in all mice by 48 h post‐injection. Severe but again reversible anatomopathological changes are also observed: massive cellular depletion, necrosis and edema of lymphoid organs, leakage syndrome and inflammatory cell infiltrates of the lung, cell vacuolization, necrosis and vascular congestion of the liver.

All these data are similar to the clinical and immunological manifestations of the OKT3‐induced reaction in patients and, thus, provide an invaluable experimental tool to study its mechanisms and explore its prevention.
Sequential appearance of γ/δ‐ and α/β‐bearing T cells in the peritoneal cavity during an i.p. infection with Listeria monocytogenes
Authors: Ohga Shouichi , Yoshikai Yasunobu , Takeda Yasuyuki , Hiromatsu Kenji , Nomoto Kikuo
Published: March 2007

To search for a potential role of T cell antigen receptor (TcR) γ/β‐bearing cells in host‐defense against Listeria monocytogenes, we analyzed the sequential appearance of γ/δ and α/β T cell in the peritoneal exudate cells (PEC) during an i.p. infection with sublethal dose (2 × 103) of viable Listeria organisms in mice. The PEC on day 1 after the infection consisted of 48% macrophages and 50% lymphocytes, most of which were surface IgM+ (B) cells. The number of PEC increased to the maximal level by day 3. The PEC at this stage contained an appreciable number of CD3+ T cells in addition to a large number of macrophages. Of the CD3+ cells, the proportion of CD4−CD8− cells, most of which expressed no TcR α/β, increased to the maximal level on day 3 after the infection. In correlation with an increased number of CD3+CD4−CD8−TcR α/β− cells, high level of TcR γ/δ chain gene messages was detected in the nonadherent population of the PEC on this stage. On the other hand, the PEC on day 8 contained an increased number of CD4+CD8− and CD4−CD8+ cells which expressed TcR α/β chain on their surface. These results suggest that the γ/δ T cells precede the α/β T cells in appearance during listerial infection. The γ/β T cells may be involved at the first line of the host‐defense against Listeria.
Defective V‐to‐J recombination of T cell receptor γ chain genes in scid mice
Authors: Schuler Walter , Schuler Amelie , Bosma Melvin J.
Published: March 2007

The status of T cell receptor γ chain genes (TcR γ) in 11 spontaneous T cell lymphomas from mice with severe combined immune deficiency (scid) was analyzed. We found that as a result of large abnormal deletions accompanying attempted site‐specific V‐to‐J recombinations, 36 of 47 rearranged TcR γ genes lacked the variable (V) and/or joining (J) region gene segment involved in this attempted recombination. No such deletions were found in T cell lymphomas from normal mice. We interpret our data as indicating a defective V‐to‐J recombination in scid lymphocytes consistent with our earlier observation of faulty D‐to‐J recombination in transformed scid lymphocytes (Schuler et al., Cell 1986. 46: 963). The present results further support the hypothesis that the scid mutation affects a component of the VDJ‐recombinase system used in common by B and T cells to assemble antigen receptor genes.
In rat B lymphocyte genesis sixty percent is lost from the bone marrow at the transition of nondividing pre‐B cell to sIgM+ B lymphocyte, the stage of Ig light chain gene expression
Authors: Deenen Gerrit Jan , Van Balen Iteke , Opstelten Davine
Published: March 2007

The cycling B precursor cells in rat bone marrow (BM) that carry the B220 antigen and no surface Ig daily produce 780 million new cells. The pool of recirculating B lymphocytes in the rat, however, renew at a rate of only about 40 million cells/day. To analyze at which stages in B lymphocyte genesis the cell loss occurs, we identified post‐mitotic cells in the rat BM B lineage, and determined their renewal rates. We used 5‐bromo‐deoxyuridine (BrdUrd) to label DNA‐synthesizing cells, identifying incorporated BrdUrd with the mouse monoclonal antibody BU‐1. B lineage cell subsets were identified by the markers HIS24 antigen (rat B220), terminal deoxynucleotidyl transferase (TdT), Ig μ heavy chain, and complete Ig. By use of double and triple immunocytology, we determined the extent of BrdUrd incorporation in the various B lineage compartments [HIS24+TdT−Ig−, TdT+, cytoplasmic μ chain (cμ)+ surface (s) IgM− pre‐B, sIgM+ B]. Both sIgM+ B lymphocytes and all B precursors with cell diameters < 11–12 μm were virtually devoid of DNA synthesis, as indicated by S‐phase indices below 2%. In contrast, S‐phase indices of large B precursors ranged between 43%–66%.

We established the renewal rates of nondividing BM B lineage cells by placing osmotic minipumps containing BrdUrd subcutaneously in the flank of rats. The nondividing BM B lineage cells all renewed rapidly at rates between 2.4% and 5.6%/h, representing average half‐lives of 29 to 12 h. In absolute numbers, the renewal/day/whole body BM was 165 × 106 for sIgM+ B lymphocytes, 422 × 106 for small cμ+ sIgM− pre‐B cells, 89 × 106 for small TdT+ cells and 35 × 106 for small HIS24+TdT−Ig− cells. Assuming that recirculating B lymphocytes in the periphery are the descendants of BM sIgM+ B lymphocytes, which in their turn are the progeny of small pre‐B cells, the renewal data indicate the following. Of the 165 million potentially available BM B lymphocytes, only 40 million cells become incorporated in the pool of recirculating B lymphocytes, representing a loss of 75%. BM B lymphocytes, in turn, use only (165/422 × 100% =) 40% of the potential output from their immediate precursors. The 60% loss that occurs here may reflect the extent of aberrant Ig light chain gene rearrangement in normal B lymphocyte genesis. In the earlier stages in B lymphocyte genesis, many of the 780 × 106 new cells produced by proliferating precursors apparently do not reach the small pre‐B compartment (which takes up only 422 × 106 new cells/day). The overall loss amounts to a total of 95%, with [(780‐165)/780 × 100% =] 80% in the BM and [(165‐40)/780 × 100% =] 15% in the periphery. We discuss mechanisms potentially underlying the loss at the various stages in B lymphocyte development.
Stimulation of lymphocyte migration by a novel low‐molecular weight compound in normal human skin and plasma
Authors: Bacon Kevin B. , Fincham Nicholas J. , Camp Richard D. R.
Published: March 2007

Normal human skin contains an extravascular population of lymphocytes which have been proposed to play a central role in immunosurveillance. The hypothesis that physiologic lymphocyte chemoattractants induce the migration of these cells has been tested by analysis of samples from normal human skin by an in vitro lymphocyte migration assay in combination with high‐performance liquid chromatography (HPLC). Dilution‐related lymphocyte migration was seen on assay of aqueous extracts of stratum corneum, analysis after ultrafiltration consistently demonstrating activity in < 1‐kDa fractions. Similar results were obtained with chamber fluid from abraded normal skin and with normal plasma. A novel, polar, low‐molecular weight component in plasma and chamber fluid was purified to homogeneity by reverse‐phase HPLC. Material from the two sources possessed identical retention times on reverse‐phase HPLC and similar ultraviolet (UV) absorbance spectra (UV maximum 274 nm). The factor has been termed plasma‐associated lymphocyte chemoattractant (PALC). When purified to homogeneity, PALC induced concentration‐related lymphocyte migration, was extractable in lipid solvents and was not peptide in nature, as determined by mass spectrometric and amino acid analyses and proteolytic enzyme digestion. It is proposed that PALC may play a role in mediating physiologic margination, adherence and migration of lymphocytes in normal human skin.
Competition among class II major histocompatibility molecules for presentation of tyrosine‐azobenzenearsonate occurs in vivo and in vitro
Authors: Baskar Sivasubramanian , Leskowitz Sidney
Published: March 2007

We have compared by functional assays the relative preference among Ia molecules for their ability to present tyrosine‐azobenzenearsonate (ABA‐Tyr) to T cells. Immunization of B10. BR mice (IAk, IEk) with ABA‐Tyr resulted in the induction of IAk‐restricted T cells only. Immunization of B10.A(5R) mice (IAbb, IEb/k) gave only IEb/k‐restricted T cell clones even though IAb‐restricted responses could be induced in C57BL/6 mice (IAb). These results indicated that IAk was preferred over IEk and IEb/k was preferred over IAb for presentation of ABA‐Tyr. A comparison between IAk and IEb/k made by immunizing [B10.BR × B10.A(5R)]F1 mice (IAk, IEk, IAb, IEb/k), showed that IEb/k was favored over IAk. No IAb‐ or IEk‐restricted response was seen.

Further attempts were made to compare Ia preference for ABA‐Tyr presentation by competitive inhibition assays. It could be shown that the presence of IEb/k molecules on an accessory cell interfered with the ability of IAb molecules on the same cell to present ABA‐Tyr to an IAb‐restricted T cell clone by direct competition. Such a competition was not observed between IAk and IEk. Finally, it could be shown that addition of ABA‐Tyr inhibited the presentation of moth cytochrome‐c peptide (81–103) by IEb/k but did not influence its presentation by IEk. From these functional studies we suggest that the binding affinity of ABA‐Tyr with the Ia molecules will fall in the order: IEb/k > IAk > IAb > IEk.
Different response of TH1 cells for stimulation with anti‐CD3 antibodies
Authors: Lohoff Michael , Schmitt Edgar , Reske‐Kunz Angelika B. , Röllinghoff Martin
Published: March 2007

In this report, evidence is provided for a further subdivision of CD4+ T helper cell lines. The earlier definition of the TH1 and TH2 subtypes was confirmed by their differential response to interleukin (IL) 1. An additional subdivision of the TH1 subset was revealed when TH1 cell lines were costimulated with anti‐CD3 antibodies and IL2. The IL2‐induced proliferation of three of the resulting TH1 lines was blocked by anti‐CD3 antibodies. By contrast, no such block was observed in a fourth TH1 cell line. In all four lines anti‐CD3 triggering caused production of IL 2. The block of proliferation was reversed neither by antigen‐presenting cells nor by phorbol 12‐myristate 13‐acetate, a protein kinase C activator. In all TH2 cells, however, anti‐CD3 triggering led to IL4 production, but had only marginal effects on IL2‐induced cell proliferation.
Cross‐recognition of a mouse H‐2‐peptide complex by human HLA‐restricted cytotoxic T cells
Authors: Murray Ruth J. , Brooks Jill M. , Rickinson Alan B. , Rowe Martin
Published: March 2007

Epstein‐Barr virus (EBV)‐specific human cytotoxic T lymphocyte (CTL) lines from a virus‐immune HLA‐A11+ donor CMc produced significant lysis of transfected mouse P815 target cells (H‐2d background) expressing an introduced HLA‐A11 heavy chain gene and the EBV gene encoding latent membrane protein (LMP). To identify the target epitope being recognized, HLA‐A11+ transfectants of P815/A11) were pre‐exposed to synthetic peptides corresponding to fragments of the LMP sequence and the cells then tested for lysis by CMc effectors; untransfected P815 parent cells were included in these assays as control recipients of the same peptides. Unexpectedly, we identified a peptide preparation (corresponding to LMP residues 124–137) which specifically sensitized not only P815/A11 cells but also the parental P815 cells to lysis. Four independent anti‐EBV CTL lines derived from donor CMc gave the same result and single‐cell cloning confirmed that lysis was mediated by a subset of the HLA‐A11‐restricted EBV‐specific CTL clones which dominate CMc effector populations. Two lines of evidence indicated that peptide presentation to the human CTL was occurring through H‐2Kd molecules on the surface of P815/A11 and P815 cells: (a) a monoclonal antibody reactive with H‐2Kd specifically blocked the lysis of both P815/A11 and P815 peptide‐treated targets, and (b) on an extended target cell panel, peptide‐mediated sensitization was also obtained with a second H‐2d mouse line A20.2J and with an H‐2Kd transfectant of the H‐2k line R1E. This work has therefore identified EBV‐specific HLA‐A11‐restricted human CTL which show fortuitous cross‐recognition of a synthetic peptide in the context of a mouse H‐2Kd molecule. Such potential for xenogeneic cross‐reactivity needs to be borne in mind in situations where the target specificity of human CTL is being analyzed on HLA gene‐transfected murine target lines.
Differences in specificity of “DPw2‐specific” cytotoxic T cell clones revealed with HLA‐mutant lines: Evidence that non‐DP HLA genes influence recognition by some clones
Authors: Pérez Miguel Sánchez , Orejas Rosalia Diez , Petersen Jean W. , de Mars Robert , Shaw Stephen
Published: March 2007

Seven allospecific cytotoxic T lymphocyte (CTL) clones derived from DPw2‐specific bulk populations were characterized by three approaches to obtain a more detailed understanding of the T cell recognition of the HLA‐DPw2 molecule. All seven of the clones were DPw2 specific and indistinguishable in specificity by three approaches: (a) patterns of lysis of panels of targets from normal donors; (b) inhibition of lysis by anti‐class II monoclonal antibodies (mAb); (c) lysis of mutant lymphoblastoid B cell Lines (LCL) with isolated loss of DP2α or DP2β gene expression (as a result of selection for resistance to DPw2‐specific CTL). However, only four of the seven CTL clones (which we designate “orthodox”) lysed all mutant DPw2+ LCL tested; the other three (“heterodox”) CTL clones showed reduced or no lysis of particular LCL which expressed DPw2 but had been mutagenized and selected for loss of DR expression. Cold target blocking experiments with the mutant LCL confirmed differences in: (a) specificity among CTL clones and (b) CTL‐defined phenotype among serologically indistinguishable DR−DPw2+ mutant LCL. Differences were not explained by different levels of DP expression by the mutant LCL. Given the nature of the mutagens and mutations, it is highly improbable that point mutations in DP account for differences in recognition. These data suggest that non‐DP HLA genes influence recognition by some DP‐specific clones, potentially due to corecognition of HLA‐DRα or another non‐DP HLA product in the context of a DPw2α/β dimer.
An exact comparison between the efficiency of two‐ and three‐cell‐type clusters in mediating helper activity
Authors: Avrion Mitchison N.
Published: March 2007

Adoptive transfer of anti‐H‐2Db‐primed B cells combined with anti‐minor histocompatibility antigen‐primed T cells enables the effect of linked and unlinked alloantigen to be compared. Over an extensive dose range the unlinked antigen proved ineffective. In a previously described, exactly comparable system for assessing the same effect on cytotoxic T cell precursors, low doses of linked antigen had proved approximately equally effective, but high doses of unlinked antigen had proved effective. This contrast supports the hypothesis that whereas T‐B collaboration is mediated by two‐cell‐type clusters, collaboration between T cells requires formation of three‐cell‐type clusters.