bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential activation requirements associated with stimulation of T cells via different epitopes of CD3
Authors: Yang  , Parkhouse
Published: March 2003

A panel of monoclonal antibodies directed to different epitopes of porcine CD3 were employed to investigate stimulation requirements of porcine T lymphocytes. It was found that epitope specificity was an important property of the anti‐CD3 antibodies that determined the requirements for T‐cell proliferation. Thus, T‐cell proliferation induced by triggering different CD3 epitopes showed three different requirements: (a) proliferation induced by the most insensitive epitope required both epitope ligation and some unknown additional signal(s); (b) proliferation induced by the most common epitopes only required epitope ligation, either by monocytes or by immobilization; (c) proliferation induced by the most sensitive epitope required neither epitope ligation nor participation of antigen‐presenting cells (APC). These findings may help to explain the previous confusion over the requirements for T‐cell activation through the CD3 pathway. Finally, the above conclusions apply only to αβ T cells, as porcine γδ T cells, either in bulk culture or isolated, did not proliferate in response to anti‐CD3 stimulation. Therefore, the mechanism underlying γδ T‐cell activation may be different from that of αβ T cells.
Human monoclonal antibodies encoded by the V4‐34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy‐chain variable region
Authors: Thorpe  , Turner  , Stevenson  , Spellerberg  , Thorpe  , Natvig  , Thompson
Published: March 2003

We have characterized the reactivities of a panel of V4‐34‐encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4‐34‐encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4‐34‐encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4‐34‐encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti‐D and the other V4‐34‐encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy‐chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.
Early cytokine responses during intestinal parasitic infections
Authors: ISHIKAWA  , GOYAL  , MAHIDA  , LI  , WAKELIN
Published: March 2003

Infections with gastro‐intestinal nematodes elicit immune and inflammatory responses mediated by cytokines released from T‐helper type‐2 (Th2) cells. In vitro assays of cells from the mesenteric lymph nodes (MLN) of experimentally infected rodents confirm that, after about 1 week, the dominant cytokine responses to mitogens and antigens are those associated with this Th‐cell subset. Polarization of the Th response in this way implies an initial local cytokine enviroment that favours Th2 development. However, experimental infections with Trichinella spiralis and Nippostrongylus brasiliensis show that, within 2 days of worms reaching the intestine, MLN cells (MLNC) respond with a Th1 rather than a Th2 response [i.e. there is an increase in mRNA for the type 1 cytokine interferon‐γ (IFN‐γ), and mitogen‐stimulated MLNC release IFN‐γ rather than interleukin‐5 (IL‐5)]. Antigen stimulation at this time does not elicit IFN‐γ release and the MLNC cannot adoptively transfer immunity. Within a few days the MLNC phenotype changes. There is a Th2 response (IL‐5 release) to both mitogen and antigen stimulation and MLNC can adoptively transfer immunity. Early release of IFN‐γ is T‐cell dependent, with CD4+ T cells playing the major role. The data are discussed in relation to factors regulating the mucosal response to invasion by parasites.
Low antigen dose favours selection of somatic mutants with hallmarks of antibody affinity maturation
Authors: GONZÁLEZ‐FERNÁNDEZ  , MILSTEIN
Published: March 2003

The immunization schedule is critical for the derivation of high‐affinity antibodies, low antigen dose being particularly favourable for the development of a more efficient memory response. To analyse the molecular events underpinning this preference, we analysed the early maturation of the response to the hapten 2‐phenyloxazolone (phOx) using low and high doses of immunogen. The phOx response is initially dominated by antibodies expressing the VkOx1‐Jk5 light chain and the hallmark of the early stages of maturation is the substitution of His 34 by Asn or Gln increasing affinity 10‐ or eightfold, respectively, and of Tyr 36 by Phe. High‐affinity antibodies express mutations at both sites. We cloned and sequenced VkOx1‐Jk5 light chains from antigen‐specific B cells taken 14 and 21 days after immunization with high and low antigen doses. We found that overall, the derived sequences were more mutated both at longer times and at higher dose. At day 14, His 34 was more frequently mutated at the higher than at the lower dose, while at day 21 the reverse was true. On the other hand, the His 34/Tyr 36 mutation pair was more frequent at low than high doses at both 14 and 21 days. Furthermore, at both times, the low immunization protocol yielded double mutants in cells with a lower mutation background. It appears therefore that while the higher dose may favour the acquisition of individual critical mutations, low‐dose immunization favours the selection of a more focused mutational pattern, whereby advantageous mutations are associated with a low mutational background.
Injection of T‐cell receptor peptide reduces immunosenescence in aged C57BL/6 mice
Authors: LIANG†  , ZHANG  , INSERRA  , JIANG  , LEE  , GARZA  , MARCHALONIS  , WATSON
Published: March 2003

Previous studies established that retrovirally infected young mice produced large amounts of autoantibodies to certain T‐cell receptor (TCR) peptides whose administration diminished retrovirus‐induced immune abnormalities. C57BL/6 young (4 weeks) and old (16 months) female mice were injected with these same synthetic human TCR Vβ8.1 or 5.2 peptides. Administration of these autoantigenic peptides to old mice prevent immunosenesence, such as age‐related reduction in splenocyte proliferation and interleukin‐2 (IL‐2) secretion. TCR Vβ peptide injection into young mice had no effect on T‐ or B‐cell mitogenesis and IL‐4 production while modifying tumour necrosis factor‐α (TNF‐α), IL‐6, and interferon‐γ (IFN‐γ) secreted by mitogen‐stimulated spleen cells. TCR Vβ injection also retarded the excessive production of IL‐4, IL‐6 and TNF‐α induced by ageing. These data suggest that immune dysfunction and abnormal cytokine production, induced by the ageing process, were largely prevented by injection of selected TCR Vβ CDR1 peptides.
From sentinel to messenger: an extended phenotypic analysis of the monocyte to dendritic cell transition
Authors: WOODHEAD  , BINKS  , CHAIN  , KATZ
Published: March 2003

The transitional stages in the relationship between sentinel monocytes and messenger dendritic cells that are active in adaptive immunity, are, as yet, unclear. To explore these events, 2‐hr adherent peripheral blood mononuclear cells were used either as monocytes, or cultured for 7 days with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) to generate dendritic cells, and the phenotypic features and relationship of the two cell populations was investigated using an extensive panel of monoclonal antibodies (mAbs). The features of the shift from monocyte to dendritic cell were also examined by daily phenotyping during the 7‐day culture period. Twenty‐five mAbs, most of which recognized known CD molecules, bound both monocytes and dendritic cells equally, whereas 19 mAbs exhibited differential staining. Four molecules not previously reported on dendritic cells were documented: CD87, CD98, CD147 and CD148. Seven cell‐surface molecules (HLA‐DQ, CD1a, CD13, CD30, CD43, CD63 and CD86) were expressed either at very low levels or not at all on monocytes, but had a strikingly increased expression on dendritic cells, suggesting a role in antigen presentation. The kinetics of monocyte to dendritic cell transition revealed a rapid activation phase within the first 24 hr, with a considerable increase in expression of the activation markers HLA‐DR, CD13, CD14 and CD98; this was followed by a down‐regulation of CD14 and a more gradual development of the other dendritic cell features over the remaining 6 days, with steady increases in CD1a, CD18, CD43, CD86, HLA‐DR and HLA‐DQ. Thus, these studies have demonstrated four novel components of the dendritic cell, and have documented the dynamic multistep nature of the process whereby an antigen‐presenting dendritic cell phenotype may emerge from a monocyte precursor.
Disease‐protected major histocompatibility complex Ea‐transgenic non‐obese diabetic (NOD) mice show interleukin‐4 production not seen in susceptible Ea‐transgenic and non‐transgenic NOD mice
Authors: BRENDEN  , BÖHME
Published: March 2003

The non‐obese diabetic (NOD) mouse is an animal model for insulin‐dependent diabetes that has many similarities to the human disease. NOD mice transgenic for the Ea gene, allowing expression of the E molecule, are protected from diabetes and rarely develop insulitis. An Ea transgene mutated in the promoter region, (ΔY) lacks E expression on most B cells, thymic medullary epithelium and primary antigen‐presenting cells, and confers no protection whatsoever. We have used these transgenic NOD mice, together with non‐transgenic NOD mice, to study the correlation of E expression and production of interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ). We show that protected E‐transgenic NOD mice have elevated levels of IL‐4 compared with non‐transgenic mice, both in the thymus and in the periphery. However, susceptible ΔY‐transgenic mice have elevated thymic IL‐4 levels, but express almost as little IL‐4 as non‐transgenic NOD mice in the periphery. This drop in peripheral IL‐4 production seen in ΔY‐transgenic mice thus correlates with the decreased E expression in the periphery of ΔY‐transgenic NOD mice. In contrast, there were no differences in IFN‐γ production between the three NOD lines. We suggest that Ea‐transgenic NOD mice have E‐selected regulatory T cells producing IL‐4, which are subsequently activated by E‐expressing primary antigen‐presenting cells in the periphery. This activation would then be instrumental for the E‐mediated protection from disease in NOD mice. Such a process would explain the total absence of protection in ΔY‐transgenic NOD mice, despite their widespread E expression.
Immunosuppressant deoxyspergualin induces apoptotic cell death in dividing cells
Authors: ODAKA  , TOYODA  , NEMOTO
Published: March 2003

Deoxyspergualin (DSG) has been found to have an antitumour and immunosuppressive activity. However, the precise mechanism of action of DSG has not been clarified. We have used its analogue, methyldeoxyspergualin (MeDSG) for in vitro culture studies of DSG since it shows good stability in aqueous solution and retains strong immunosuppressive activity. In the present study, we found that MeDSG inhibited proliferation of rapidly dividing murine T‐cell hybridomas, resulting in cell death. The cell death was accompanied by chromatin condensation and DNA cleavage at the linker regions between nucleosomes. Furthermore, MeDSG induced a reduction in mitochondrial transmembrane potential. When murine thymocytes were treated with MeDSG for 48 hr, a slight increase of DNA fragmentation was constantly observed, and selective depletion of CD4− CD8− cells was noticed. In contrast, CD4+ CD8+ cells were hardly affected. Moreover, splenic T‐cells are resistant to MeDSG‐induced apoptosis, as evaluated by measuring DNA cleavage. Our findings may account for the immunosuppressive and antitumour properties of DSG which were described in a number of previous studies.
Unresponsiveness following immunization with the T‐cell‐independent antigen dextran B512. Can it be abrogated?
Authors: SVERREMARK  , FERNANDEZ
Published: March 2003

The bacterial carbohydrate dextran B512 is a thymus‐independent (TI) antigen and a poor immunogen. Humoral responses consist primarily of IgM and no memory response is observed; rather, secondary responses to native dextran are similar to or suppressed compared with primary responses. However, immune responses to dextran can be enhanced. In this study we have used a protein–dextran conjugate that elicits a thymus‐dependent (TD) immune response against dextran. Furthermore, we used the potent adjuvant cholera toxin (CT) for the dextran immunizations. This enables us to re‐evaluate the phenomenon of poor secondary response to dextran and whether it can be abrogated. We show that native dextran‐primed mice were not able to mount IgG anti‐dextran antibody responses after repeated immunizations with the TD, protein–dextran conjugate. This was also apparent in the spleen, where almost no dextran‐specific germinal centres were detected. However, the anti‐protein antibody response was normal in these mice, demonstrating that it is only the anti‐dextran‐responding cells that are affected. The effect of CT adjuvant on these events was also evaluated. CT enhanced the humoral IgM anti‐dextran responses as well as the splenic responses to dextran. But, the isotype profile was not altered, still no IgG was produced. In contrast, mice primed with the TD conjugate and repeatedly re‐immunized with native, TI, dextran generated IgG anti‐dextran responses. Our results indicate that it is probable that the lack of proper costimulation in the initiation of the response to dextran causes the suppressed secondary dextran responses. Furthermore, these results suggest that TI and TD forms of dextran activate the same type of B cells, since TI dextran‐priming abrogated TD dextran IgG responses. The importance of the priming event for the induction of a classical memory response to carbohydrate antigens and the implications for vaccination strategies, are discussed.
Mechanism of mercury‐induced autoimmunity: both T helper 1‐ and T helper 2‐type responses are involved
Authors: HU  , MÖLLER  , ABEDI‐VALUGERDI
Published: March 2003

Mercury can induce a systemic autoimmune disease in susceptible mouse strains. H‐2s mice are particularly susceptible to mercury‐induced autoimmunity and other mouse strains are more or less resistant. T helper 1/T helper 2 (Th1/Th2) dichotomy has been proposed for resistance or susceptibility, respectively. In the current study we show that mercury treatment induced a full autoimmune response in both C57BL/6 (H‐2b) wild‐type and interleukin‐4 (IL‐4)‐deficient mice. Antibody production of all isotypes were induced, except that in IL‐4‐deficient mice there was no immunoglobulin E (IgE) and very low levels of immunoglobulin G1 (IgG1) antibody synthesis. Autoantibodies of different specificities were produced. The granular pattern of all IgG subclasses deposits were detected in the kidneys. In contrast to mercury‐treated H‐2s seconds mice, we did not detect any anti‐nucleolar autoantibodies in the sera of mercury‐treated wild‐type or IL‐4‐deficient mice. To further explore the role of Th1/Th2 cytokines in the mercury model, we performed anti‐interferon‐γ antibody treatment in IL‐4‐deficient mice together with mercury treatment and found that the production of IgG2a and IgG3, but not IgG2b, antibodies was downregulated. This indicated that besides Th2‐type cytokines, Th1‐type and other cytokines were involved as well in mercury‐induced autoimmune response. Thus, C57BL/6 mice with H‐2b genotype are highly susceptible to mercury‐induced autoimmunity, and the genetic susceptibility to mercury involves more than a predisposition of a Th1‐or Th2‐type response.
Distinct roles for transforming growth factor‐β2 and tumour necrosis factor‐α in immune deviation elicited by hapten‐derivatized antigen‐presenting cells
Authors: HECKER‡  , NIIZEKI§  , STREILEIN
Published: March 2003

The role of antigen‐presenting cells (APC) in the induction of antigen‐specific unresponsiveness was examined, using two functionally distinct murine macrophage hybridomas, #59 and #63 cells. Derivatized with the hapten (dinitrofluorobenzene; DNFB), #59 cells induced contact hypersensitivity (CH) in mice. Hapten‐derivatized #63 cells failed to induce CH. Instead, they prevented recipients from acquiring CH when exposed subsequently to a sensitizing dose of the hapten. Similarly, hapten‐derivatized #59 cells, pretreated in vitro with transforming growth factor‐β2 (TGF‐β2) lost their capacity to evoke CH, and induced tolerance. Hapten‐derivatized #63 cells and TGF‐β2‐treated #59 cells eliminated CH in mice sensitized to hapten. Reverse transcription–polymerase chain reaction analysis of mRNAs for various accessory molecules important in T‐cell activation revealed that #63 and TGF‐β2‐treated #59 cells differed only in their expression of tumour necrosis factor‐α (TNF‐α) mRNA. The latter expressed higher levels of TNF‐α mRNA than did untreated #59 cells. As a consequence, #63 and TGF‐β2‐treated #59 cells, both of which induce tolerance, secrete TNF‐α protein unlike untreated #59 cells, which do not induce tolerance to hapten. Since neutralizing anti‐TNF‐α antibodies abrogated the tolerogenic potential of #63 cells in vivo, we conclude that TGF‐β2 equips hapten‐bearing APC with the capacity to evoke systemic immune deviation in which CH is selectively silenced. We speculate that one effect of TGF‐β2 is to cause APC to up‐regulate TNF‐α production. In turn, this cytokine biases the functional property of responding hapten‐specific T cells in a direction that not only interferes with acquisition, but suppresses induction of CH.
Intrathymic function of the human cortical epithelial cell surface antigen gp200‐MR6: single‐chain antibodies to evolutionarily conserved determinants disrupt mouse thymus development
Authors: PALMER  , CROMPTON  , MARANDI  , GEORGE  , RITTER
Published: March 2003

The mouse monoclonal antibody MR6 recognizes a 200 000 MW protein (gp200‐MR6), which is expressed highly on human thymic cortical epithelial cells. The antigen is also expressed on some epithelial tumours and we have previously shown that MR6 inhibits the proliferation of the colon carcinoma cell lines HT29. However, the role of this molecule in the thymus is not known. In order to generate reagents that could be used in murine thymic functional studies we isolated antibodies specific to human gp200‐MR6, using a phage display library expressing single‐chain (sFv) antibodies. Three independent clones were isolated by panning with purified protein and their specificity was confirmed by immunohistochemistry, Western blotting and flow cytometry. In addition to human thymus, these phage antibodies also recognized the homologous antigen in mouse, pig and other species. Expressed as soluble sFv one of these clones inhibited the proliferation of HT29 cells and a mouse thymic epithelial cell line, suggesting that this antibody exhibits similar functional activity to MR6. In fetal thymic organ culture, thymocytes recovered from thymic lobes cultured in the presence of this sFv, were reduced in number fivefold compared with the control and the majority remained at the double‐negative stage of development. These data indicate that gp200‐MR6 plays an important role in thymocyte development. In addition, this is the first report to demonstrate that specific sFv can be used to study, and alter, thymic development. This work also highlights the advantage of phage antibody technology in selecting such reagents for functional assays.
T cells with a quiescent phenotype (CD45RA+) are overabundant in the blood and involuted lymphoid tissues in wasting protein and energy deficiencies
Authors: WOODWARD  , HILLYER  , HUNT
Published: March 2003

The objective of this investigation was to determine the influence of distinct forms of acute weight loss on the expression of the quiescence marker, CD45RA, by T cells in several lymphoid compartments including the blood. Male and female weanling mice, CBA/J and C57BL/6J strains, were allocated to the following groups: ad libitum intake of a complete purified diet; restricted intake of the complete diet; and ad libitum intake of an isocaloric low‐protein diet. The restricted intake protocol produced weight loss through energy deficiency (marasmic‐type malnutrition), whereas the low‐protein diet caused wasting through inadequate protein nitrogen and induced a condition mimicking incipient kwashiorkor. In one experiment, weanling mice of both strains were maintained for 14 days according to each of these dietary protocols and, in a supplementary study, C57BL/6J weanlings consumed either the complete diet or the low‐protein diet ad libitum for 21 days. Zero‐time control groups (19‐days old and 23‐days old in C57BL/6J and CBA/J strains, respectively) were included in the first experiment to control for ontogeny‐related change. Expression of CD45RA was assessed by two‐colour flow cytometry in CD4+ and CD8+ T cells from the spleen, mesenteric lymph nodes and blood. Within 14 days, energy‐restricted mice exhibited a high percentage of CD4+ T cells expressing CD45RA in all three lymphoid compartments in both mouse strains (an average of 50% CD45RA+ versus 9% in well‐nourished controls), and a similar outcome was apparent in the CD8+ subset (93% CD45RA+ versus 63%). Mice fed the low‐protein diet required up to 21 days to exhibit the same imbalance within the CD4+ T‐cell subset (33% CD45RA+ versus 4% in well‐nourished controls). A shift toward a quiescent phenotype occurs throughout the peripheral T‐cell system in acute wasting disease. Consequently, the quiescence‐activation phenotype of blood T cells reflects the same index in secondary lymphoid organs in such pathologies. Naive‐type quiescence among T cells is implicated as a component of depressed adaptive immunocompetence in the advanced stages of diverse forms of acute weight loss.
Characterization of a surface membrane molecule expressed by natural killer cells in most inbred mouse strains: monoclonal antibody C9.1 identifies an allelic form of the 2B4 antigen
Authors: KUBOTA  , KATOH  , MUGURUMA  , KOYAMA
Published: March 2003

A newly generated monoclonal antibody (mAb C9.1) described in this study identifies a surface membrane molecule that is involved in the lytic programme of activated natural killer (NK) cells. This conclusion is based on the facts that, first, this antigen was expressed on the vast majority of surface immunoglobulin (sIg)− CD3− CD4− CD8− spleen lymphocytes, albeit it was also present on minor subsets of sIg+ B (≈7%) and CD3+ T (≈2%) lymphocytes; second, that all splenic NK activity was contained within the C9.1+ cell population, and was almost totally abolished by treatment of spleen cells with mAb C9.1 and complement; third, that mAb C9.1 was capable of increasing interleukin‐2‐cultured and in vivo polyinosinic:polycytidylic acid‐activated, NK cell‐mediated, antibody‐redirected lysis, but not freshly isolated NK cell‐mediated killing. Furthermore, the strain distribution of the C9.1 antigen was shown to be antithetical to that of the 2B4 antigen already described as a molecule associated with major histocompatibility complex‐unrestricted killing mediated by activated NK cells. The gene encoding C9.1 antigen was linked to the Akp1 isozyme locus on chromosome 1 close to the 2B4 gene. Although C9.1 and 2B4 were monomeric glycoproteins of 78 000 MW and 66 000 MW, respectively, removal of N‐linked sugars from both antigens by endoglycosidase F yielded identical protein backbones of 38 000 MW. Thus, all of these results suggest that mAb C9.1 recognizes an allelic form of the 2B4 antigen. However, the detection of mAb C9.1‐reactive antigen on a minor subset of B cells may suggest a possible reactivity of mAb C9.1 with some product of other members of the 2B4 family genes.
Phenotypic analysis of local cellular responses in calves infected with bovine respiratory syncytial virus
Authors: MCINNES  , SOPP  , HOWARD  , TAYLOR
Published: March 2003

Changes in lymphocyte subsets in the trachea, pulmonary tissue, bronchoalveolar lavage (BAL), peripheral blood and bronchial lymph node (BLN) of gnotobiotic calves infected with bovine respiratory syncytial virus (BRSV) were analysed by flow cytometry. Following BRSV infection, virus titres in the nasopharynx reached a peak between days 5 and 7 and infection was resolving from day 10. Although calves did not develop signs of clinical respiratory disease, there was evidence of gross pneumonia and histological changes typical of BRSV bronchiolitis, which were most extensive from day 7–10 of infection. Following BRSV infection there was a recruitment of CD8+ T cells into the trachea and lung, which peaked on day 10 after infection. Thus, there were approximately equal numbers of CD8+ and CD4+ T cells in the lung and trachea of uninfected calves, whereas by day 10 of infection, CD8+ cells outnumbered CD4+ cells by 3:1 in the lungs and 6:1 in the trachea of the infected calves. Although the increase in CD4+ T cells into the lungs was less marked than that of CD8+ T cells, changes in expression of CD45R, CD45RO, l‐selectin and interleukin‐2 receptors all suggested that CD4+ T cells were activated during BRSV infection. Changes in γδ T cells were not observed in BRSV‐infected calves. There was a marked increase in B cells in the BLN after infection and BLN CD4+ T cells changed from the majority expressing l‐selectin and CD45R in uninfected calves to a predominance of l‐selectin− CD45R− CD45RO+ phenotype, 10 days after infection. In conclusion, CD8+ T cells constitute the major lymphocyte subpopulation in the respiratory tract of calves recovering from BRSV infection.
Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP)
Authors: PÉREZ DE LA LASTRA  , VAN DEN BERG  , BULLIDO  , ALMAZÁN  , DOMÍNGUEZ  , LLANES  , MORGAN
Published: March 2003

Pig membrane cofactor protein (MCP; CD46) is a 50 000–60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I‐mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane‐bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane‐bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross‐reactive with human MCP and only one showed cross‐reactivity with leucocytes from a panel of large mammals – a weak cross‐reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP‐catalysed C3 degradation by factor I.
Inability of interleukin‐12 to modulate T‐helper 0 effectors to T‐helper 1 effectors: a possible distinct subset of T cells
Authors: HU  , SALGAME
Published: March 2003

Interleukin‐12 (IL‐12) strongly favours the development of T‐helper 1 (Th1)‐type cells through its ability to induce interferon‐γ (IFN‐γ) production by natural killer cells and T cells. In the present work we analysed the effects of IL‐12 on the synthesis and secretion of IFN‐γ and IL‐4 by human T‐cell clones. Several previously described human T‐cell clones exhibiting Th1, Th2 or Th0 phenotypes were used for these analyses. We demonstrated, by enzyme‐linked immunosorbent assay (ELISA) and intracytoplasmic staining, that, in Th0 clones, IL‐12 up‐regulated the production of both IFN‐γ and IL‐4 and was unable to modulate these cells to Th1‐type. The up‐regulation of cytokine gene expression was transcriptionally regulated and was not due to differences in mRNA stability. In Th1 cells, IL‐12 up‐regulated only IFN‐γ and not IL‐4. However, in Th2 cells, both IFN‐γ and IL‐4 were up‐regulated by IL‐12. This suggests that Th2 cells may be less stable than Th1 cells. We also observed that human Th2 cells expressed the IL‐12β2 receptor, in contrast to murine Th2, which lacks this receptor. The observed differences in the effects of IL‐12 on the three T‐cell subsets may have important ramifications for IL‐12‐based therapies.
The expansion of human γδ T cells in response to Daudi cells requires the participation of CD4+ T cells
Authors: Fayen  , Tykocinski
Published: March 2003

The Burkitt’s lymphoma cell line Daudi is a potent inducer of human γδ T‐cell expansion. Using an in vitro culture system comprised of irradiated Daudi cells as stimulators and normal human lymphocytes as responders, the cellular determinants of this response were investigated. Three of four monoclonal antibodies (mAbs 1‐1C4, L243, and 9.3F10) directed against disparate epitopes of human major histocompatibility complex (MHC) class II, as well as a mAb with specificity for CD4 (OKT4), inhibited the expansion of γδ T cells in response to Daudi cell stimulators. mAbs with a specificity for CD74 and CD8 were non‐inhibitory. Lymphocyte depletion experiments demonstrated a critical role for the CD4+ T‐cell subset in the expansion of γδ T cells. Other data pointed towards requirements for direct cell contact in this system, and the addition of exogenous recombinant interleukin (IL)‐2, IL‐4, and IL‐12 failed to reconstitute γδ T‐cell expansion in CD4+ lymphocyte‐depleted cultures. These results complement previous findings in murine infectious disease and mycobacterial systems, providing a direct demonstration that CD4+ T cells play a role in γδ T‐cell expansion through an interaction with human leucocyte antigen (HLA) class II on Daudi cells. The data point towards important functional links between the acquired and natural immune systems.
Tissue distribution of the rat analogue of decay‐accelerating factor
Authors: Spiller  , Hanna  , Morgan
Published: March 2003

In humans, decay‐accelerating factor (DAF) is a widely distributed, cell‐bound inhibitor of the complement activation enzymes and plays a key role in regulating complement activation, preventing the generation of anaphylotoxins and opsonins, and protecting against complement‐mediated lysis. Rodent analogues of DAF have recently been identified, providing a new avenue for the analysis of function. Rat DAF was cloned in our laboratory. Here we describe the generation of monoclonal antibodies (mAbs) against rat DAF, using transfected cells as immunogen, and their use in the analysis of the distribution of DAF in the rat by flow cytometry, Western blot analysis and immunohistochemistry. One of the mAbs was found to block the complement inhibitory function of rat DAF, offering the prospect of neutralization of DAF function in vivo. The antibodies have also been used for purification of DAF from rat erythrocytes by affinity chromatography. Rat DAF purified in this manner was similar in molecular mass to human DAF. The purified protein incorporated into lipid membranes, confirming the presence of a glycolipid anchor, and incorporated protein strongly inhibited the rat C3 convertase. Rat DAF was strongly expressed on endothelia throughout the animal and was also present in most tissues and organs. DAF expression was weak or absent in the brain and on circulating and spleen‐resident T cells. Strong DAF expression observed in the kidney was restricted to the glomerulus and Bowman’s capsule. DAF expression in the testis was found only in association with the later stages of spermatogenesis.
UVB exposure‐induced systemic modulation of Th1‐ and Th2‐mediated immune responses
Authors: Garssen  , Vandebriel  , De Gruijl  , Wolvers  , Van Dijk  , Fluitman  , Van Loveren
Published: March 2003

Exposure to ultraviolet light, especially UVB wavelengths, can impair immune responses in animals and humans. It is remarkable that this immunomodulation is not restricted to the exposed skin but is also found at other sites, i.e. systemic (distant) immunosuppression. A frequently proposed hypothesis is that UVB exposure inhibits, specifically, T helper 1 (Th1)‐mediated immune responses. The major reason for this is that contact hypersensitivity (CHS) and delayed‐type hypersensitivity (DTH), both Th1‐mediated immune responses, are very sensitive to UVB. For this reason these models are frequently used for photoimmunology studies. In the present study, the effects of UVB exposure were investigated in classical models for Th1‐mediated immunity, i.e. CHS models in which picrylchloride or oxazolone were used as low‐molecular‐weight chemical antigens. In these models, CHS responsiveness and cytokines were measured, the latter by both reverse transcription–polymerase chain reaction (RT–PCR) and enzyme‐linked immunosorbent assay (ELISA). The CHS responses to both contact sensitizers (picrylchloride and oxazolone) were suppressed significantly by pre‐exposure to repeated suberythemal UVB exposure. Interferon‐γ (IFN‐γ), interleukin (IL)‐12 and IL‐4, but not IL‐10, were detectable in spleen and draining lymph nodes of sensitized BALB/c mice. Repeated UVB exposure prior to sensitization at a distant locus inhibited both IFN‐γ and IL‐12 but not IL‐4. In BALB/c mice sensitized with ovalbumin (OVA) in the absence of complete Freund’s adjuvant, a model for Th2‐mediated immunity, OVA‐specific serum IgE and cytokine profiles in the spleen were analysed. Sensitization did lead to a significant increase in OVA‐specific IgE serum titres. Pre‐exposure to UVB resulted in a decreased OVA‐specific IgE serum titre. Both RT–PCR and ELISA showed increased levels of IFN‐γ, IL‐4 and IL‐10 in the spleens of OVA‐sensitized mice. The production of IFN‐γ and IL‐4 was not affected by UVB pre‐exposure. In contrast, the production of IL‐10 was significantly increased. This was probably caused by an up‐regulation of Th2 cells. It is remarkable that IFN‐γ is significantly suppressed by UVB in Th1‐mediated immune reactions but not in Th2‐mediated immune reactions where it even appears to increase. IL‐10, which is up‐regulated by UVB pre‐exposure and produced by, among others, Th2 cells, may represent a shift from Th1‐ to Th2‐mediated immune mechanisms. However, IL‐10 can also inhibit Th2 responses, which might be the reason for a decreased IgE titre in the Th2 model. From the results of this study it is concluded that UVB exposure prior to sensitization/immunization not only inhibits Th1‐mediated but also Th2‐mediated immune responses.
Interferon‐γ‐ and interleukin‐4‐targeted gene therapy for atopic allergic disease
Authors: Kang  , Kim  , Kim
Published: March 2003

Two cytokines, interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4), which play critical roles in the regulation of serum IgE level by directing the interplay of T helper (Th)1 and Th2 cells, were chosen as targets for gene therapy. Anti‐allergic activity was evaluated by determining the serum IgE level, and the functional status of each helper T cell was monitored by the serum concentrations of IgG1 and IgG2a. Experimental animals (BALB/c mice) were divided into four groups: the control group; the ovalbumin (OVA) group; the IFN‐γ group; and the IL‐4 group. The control group was injected with saline and the OVA group with OVA–alum. The IFN‐γ and IL‐4 groups were treated with OVA–alum plus the cDNAs of mouse IFN‐γ and IL‐4 in an expression vector. These treatments were applied intramuscularly on a monthly basis for 4 months. OVA–alum treatment significantly increased the serum IgE and IgG1 concentrations, but did not affect IgG2a. Concomitant treatments with the cDNA of IFN‐γ or IL‐4 returned the serum IgE almost to the control level and significantly suppressed the OVA‐induced increase of IgG1. IFN‐γ cDNA increased the serum IgG2a but IL‐4 cDNA had no affect. These results suggest that IFN‐γ inhibited the OVA‐induced IgE production by suppressing the Th2 pathway and by enhancing the Th1 pathway. Administration of IL‐4 cDNA suppressed the OVA‐induced enhancement of IgE production by inhibiting the Th2 pathway rather than by potentiating it.
The human high‐affinity immunoglobulin G receptor activates SH2‐containing inositol phosphatase (SHIP)
Authors: Cameron  , Allen
Published: March 2003

On cytokine‐primed U937 cells, aggregation of the human high‐affinity immunoglobulin receptor, FcγRI, initiates signal transduction cascades which lead to the release of calcium from intracellular stores and no significant calcium influx. In these cells, aggregation of FcγRI results in no significant increase in inositol trisphosphate production, but rather phospholipase D is activated. Here we show that, in interferon‐γ (IFN‐γ)‐primed cells, the SH2 containing inositol 5′ phosphatase, SHIP, is constitutively associated with the membrane fraction. Following aggregation of FcγRI, SHIP is rapidly and transiently tyrosine phosphorylated and becomes associated with the adapter molecule Shc. Shc also becomes tyrosine phosphorylated and translocates from the cytoplasm to the membrane fraction concomitant with the association between Shc and SHIP. Further, SHIP and Shc appear to be recruited to membrane‐associated immune complexes following FcγRI aggregation. As no immunoreceptor inhibitory motif has been demonstrated to associate with FcγRI, these data suggest that SHIP may be recruited to the receptor through an SH2 domain interaction with Shc.
Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat‐labile enterotoxin
Authors: De Haan  , Holtrop  , Verweij  , Agsteribbe  , Wilschut
Published: March 2003

The Escherichia coli heat‐labile enterotoxin (LT) is an exceptionally effective mucosal immunogen and mucosal immunoadjuvant towards coadministered antigens. Although, in general, the molecular basis of these properties is poorly understood, both the toxic ADP‐ribosylation activity of the LTA subunit and the cellular toxin receptor, ganglioside, GM1‐binding properties of the LTB‐pentamer have been suggested to be involved. In recent studies we found that GM1‐binding is not essential for the adjuvanticity of LT, suggesting an important role for the LTA subunit in immune stimulation. We now describe the immunomodulatory properties of recombinant LTA molecules with or without ADP‐ribosylation activity, LTA(His)10 and LTA‐E112K(His)10, respectively. These molecules were expressed as fusion proteins with an N‐terminal His‐tag to allow simple purification on nickel‐chelate columns. Their immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen‐specific serum immunoglobulin‐isotype and ‐subtype responses and mucosal secretory immunoglobulin A (IgA) responses were monitored using enzyme‐linked immunosorbent assay. With respect to immunogenicity, both LTA(His)10 and LTA‐E112K(His)10 failed to induce antibody responses. On the other hand, immunization with both LT and the non‐toxic LT‐E112K mutant not only induced brisk LTB‐specific, but also LTA‐specific serum and mucosal antibody responses. Therefore, we conclude that linkage of LTA to the LTB pentamer is essential for the induction of LTA‐specific responses. With respect to adjuvanticity, both LTA(His)10 and LTA‐E112K(His)10 were found to stimulate serum and mucosal antibody responses towards coadministered influenza subunit antigen. Remarkably, responses obtained with LTA(His)10 were comparable in both magnitude and serum immunoglobulin isotype and subtype distributions to those observed after coimmunization with LT, LT‐E112K, or recombinant LTB. We conclude that LTA, by itself, can act as a potent adjuvant for intranasally administered antigens in a fashion independent of ADP‐ribosylation activity and association with the LTB pentamer.
Macrophages expressing heat‐shock protein 65 play an essential role in protection of mice infected with Plasmodium yoelii
Authors: Zhang  , Hisaeda  , Sakai  , Ishikawa  , Hao  , Nakano  , Ito  , Himeno
Published: March 2003

C57BL/6 (B6) mice are resistant to infection with the non‐lethal (NL) strain of Plasmodium yoelii 17X, while being susceptible to that with the lethal (L) strain. The 65 000 MW heat‐shock protein (hsp 65) was strongly expressed in splenic adherent cells of B6 mice 10 days after infection with the NL strain of P. yoelii but only slightly in those from mice infected with the L strain. Mice which had survived infection with the NL strain were resistant to challenge with the L strain and hsp 65 was strongly expressed in splenic adherent cells of these mice. Severe combined immunodeficient mice and nude mice were susceptible to malaria infection even with the NL strain and did not express hsp 65 after infection, suggesting that T cells are required for the expression of hsp 65 as well as for protective immunity. B6 mice treated intraperitoneally with carrageenan, which impairs the macrophage function, became susceptible to NL strain infection, indicating that macrophages play an important role as the final effectors in protective immunity. These results demonstrate that the hsp 65 expressed by macrophages correlates closely with protection against P. yoelii infection.
Expression of B7.1 in a MUC1‐expressing mouse mammary epithelial tumour cell line inhibits tumorigenicity but does not induce autoimmunity in MUC1 transgenic mice
Authors: Smith  , Burchell J. M. , Graham  , Cohen  , Taylor‐Papadimitriou
Published: March 2003

The MUC1 epithelial mucin, which is overexpressed and aberrantly glycosylated in breast and other carcinomas, is also expressed on the apical surface of most normal glandular epithelial cells. Since clinical trials evaluating the efficacy of MUC1‐based vaccines have been initiated in breast cancer patients, it is important to address the question of whether an effective immune response to the cancer associated mucin can be generated without inducing autoimmunity. Since non‐classic cytotoxic T lymphocyte (CTL) responses to MUC1 have been reported, it is also relevant to examine the role of costimulatory molecules in the effective presentation of MUC1 based antigens. We have therefore looked at the effect of expressing B7.1 on the tumorigenicity of a MUC1 expressing mammary epithelial cell line (410.4) in a transgenic mouse expressing MUC1 on its normal glandular epithelial tissues. Coexpression of B7.1 with MUC1 in 410.4 cells resulted in a dramatic inhibition of tumour growth which depended on the activity of CD4+ and CD8+ T cells. The epithelial tissues in the transgenic mice able to reject the B7.1, MUC1‐expressing tumours showed no evidence of degeneration and the mice survived their normal life span. The results demonstrate that an immune response to the MUC1 antigen can be induced in MUC1 transgenic mice and that presentation of the antigen, whether directly or by cross‐priming, is markedly enhanced by coexpression of B7.1.
CD44 is involved in selective leucocyte extravasation during inflammatory central nervous system disease
Authors: Brennan F. R. , O'Neill J. K. , Allen S. J. , Butter C. , Nuki G. , Baker D.
Published: March 2003

Clinical signs of experimental autoimmune encephalomyelitis (EAE) are associated with the selective recruitment of CD4+ memory (CD45RBlow CD44high) T cells into the central nervous system (CNS). However, we have found that many of these recently recruited memory cells are CD44low, suggesting that the CD44 antigen may be involved in, and transiently lost during, the extravasation process. Indeed, administration of a CD44‐specific antibody (IM7.8.1) induced leucocyte CD44 shedding and both prevented the development and ameliorated the severity of established EAE by inhibiting mononuclear cell infiltration into the CNS. Trafficking of cells into lymph nodes, however, a property mainly of naïve cells, was essentially unaffected. In contrast, treatment with antibody to very late activation antigen‐4 (VLA‐4) prevented homing to both the CNS and to lymph nodes. This study contests previous reports that dismissed a role for CD44 in inflammation of the CNS and, coupled with observations in murine dermatitis and arthritis, suggests that CD44 is involved in the homing of primed lymphocytes to sites of inflammation. CD44 should therefore be considered a target for immunotherapy of T‐cell‐mediated inflammatory diseases, such as multiple sclerosis.
In vivo selective expansion of a tumour‐specific cytotoxic T‐cell clone derived from peripheral blood of a melanoma patient after vaccination with gene‐modified autologous tumour cells
Authors: Sun Y. , Möller P. , Berking C. , Schlüpen E.‐M. , Volkenandt M. , Schadendorf D.
Published: March 2003

Melanoma‐specific cytotoxic T lymphocytes (CTL) can be generated from peripheral blood lymphocytes (PBL) by mixed lymphocyte–tumour cell cultures. Analysis of CTL precursor frequencies in peripheral blood of melanoma patients is generally used for immunomonitoring purposes to evaluate vaccination efficacy. At present, it is unclear whether PBL‐derived CTL generated in vitro are indicative of an anti‐tumour immune response in vivo. Three tumour‐specific human leucocyte antigen (HLA)‐B/C‐restricted CTL clones were derived from peripheral blood of a melanoma patient immunized with interleukin‐7 (IL‐7) gene‐modified tumour cells. CTL clones differing in their T‐cell receptor‐γ (TCRγ) rearrangement produced interferon‐γ, IL‐4 and/or IL‐10. On the basis of their unique TCRγ gene rearrangements clone‐specific primers were generated for detection of clone‐specific DNA by polymerase chain reaction. One CTL clone (E5) of the three was found to be selectively expanded in one of seven metastases obtained at autopsy, as determined by Southern blot hybridization. However, the presence of E5 in only one of seven metastases at death indicates that the in vivo accumulation of the specific CTL clone was not sufficient to contain tumour progression. Nevertheless, our data support the proposition that analysis of anti‐tumour activity of PBL‐derived CTLs may reflect an anti‐tumour immune response in vivo.
Adhesion to fibronectin promotes the activation of the p125FAK/Zap‐70 complex in human T cells
Authors: Bearz A. , Tell G. , Formisano S. , Merluzzi S. , Colombatti A. , Pucillo C.
Published: March 2003

The β1 integrins are a family of heterodimeric adhesion receptors involved in cell‐to‐cell contacts and cell‐to‐extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, α4β1 and α5β1, can function as costimulatory molecules in T‐cell receptor (TCR)‐dependent T‐cell activation. In the current study the Jurkat T‐cell line was used as a model system to investigate the TCR‐independent role of cell adhesion to fibronectin in the activation of Zap‐70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, was induced. Moreover, clustering of fibronectin receptors led to the detection of a p125FAK/Zap‐70 complex. Finally, while the complex between fak‐B, another protein kinase localized to focal adhesion sites, and Zap‐70 was detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap‐70 complex appeared specifically induced following fibronectin‐mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the β1 integrin family mediate signalling pathways in T cells.