T cell receptor beta chain lacking the large solvent-exposed Cbeta FG loop supports normal alpha/beta T cell development and function in transgenic mice. (1/23)

The striking and unique structural feature of the T cell receptor (TCR) beta chain is the bulky solvent-exposed FG loop on the Cbeta domain, the size of almost half an immunoglobulin domain. The location and size of this loop suggested immediately that it could be a crucial structural link between the invariant CD3 subunits and antigen-recognizing alpha/beta chains during TCR signaling. However, functional analysis does not support the above notion, since transgene coding for TCR beta chain lacking the complete FG loop supports normal alpha/beta T cell development and function.  (+info)

Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules. (2/23)

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.  (+info)

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors. (3/23)

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.  (+info)

The phytoestrogen genistein suppresses cell-mediated immunity in mice. (4/23)

The soy phytoestrogen, genistein, induces thymic atrophy when administered to ovariectomized mice by injection or in the diet. Injected genistein also causes decreased humoral immunity, but the effects of genistein on cell-mediated immunity have not been addressed. Here we examined effects of injected and dietary genistein on cell-mediated immune responses. Female C57BL/6 mice (25- to 27-days-old) were ovariectomized, then placed on phytoestrogen-free feed 5 days later. Seven days after ovariectomy, they were given daily subcutaneous injections of either dimethylsulfoxide (DMSO) or genistein (8, 20, 80 mg/kg) for 28 days; some mice were given 80 mg/kg genistein plus the anti-estrogen ICI 182,780 (5 mg/kg/week). Cell-mediated immune response was tested by analyzing the delayed-type hypersensitivity (DTH) response to a hapten, 4-hydroxy-3-nitrophenyl acetyl succinimide (NP-O-SU), at the end of treatment. Reversibility of the effects of genistein was tested by measuring the DTH response in mice that were given genistein (20 or 80 mg/kg) for 28 days, then allowed to recover for 28 days. To determine if dietary genistein could affect cell-mediated immunity, mice ovariectomized as above were fed genistein at 0, 1000 or 1500 parts per million (ppm) for 28 days. There was a 46-67% decrease in the DTH response in the footpads of mice injected with 8-80 mg/kg genistein compared with controls (P<0.05 vs control for all treatment groups); these effects were reversible. On histopathological examination of the feet, there was decreased cell infiltration in genistein-treated animals compared with controls, and the numbers of CD4(+) and CD8(+) T cells in popliteal lymph nodes were reduced. The effects of genistein are mediated through both estrogen receptor (ER) and non-ER pathways, as the anti-estrogen ICI 182,780 only partially blocked the effects of genistein on the DTH response. Dietary genistein (1000 or 1500 ppm) decreased cell-mediated immunity while producing serum genistein concentrations in the physiological range for humans under certain nutritional conditions. Further work is needed to determine if dietary genistein and phytoestrogen exposure can produce effects on cell-mediated immunity in humans or other animals under various nutritional conditions.  (+info)

Metal-binding chimeric antibodies expressed in Escherichia coli. (5/23)

Metallothionein, a well-characterized biological chelator of metals, has been genetically fused to the binding domain of an antibody and expressed in the periplasm of Escherichia coli. Specific delivery of 109Cd to immobilized hapten or to haptenated cells was demonstrated directly in periplasmic extracts. This approach is potentially useful for targeted radiotherapy and diagnostic imaging. We find six to seven atoms of metal per active antigen-combining site. Absence of the Fc portion of the immunoglobulin along with low immunogenicity of metallothionein-metal complexes should reduce immunologic reactions.  (+info)

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses. (6/23)

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.  (+info)

Differential segmental flexibility and reach dictate the antigen binding mode of chimeric IgD and IgM: implications for the function of the B cell receptor. (7/23)

Mature, naive B cells coexpress IgD and IgM with identical binding sites. In this study, the binding properties of such IgM and IgD are compared to determine how size and shape may influence their ability to bind Ag and thus function as receptors. To dissect their intrinsic binding properties, recombinant IgM and IgD were produced in soluble form as monomers of the basic H(2)L(2) Ab architecture, each with two Ag binding sites. Since these sites are connected with a hinge region in IgD and structural Ig domains in IgM, the two molecules differ significantly in this region. The results show that IgD exhibited the larger angle and longer distance between its binding sites, as well as having the greater flexibility. Relative functional affinity was assessed on two antigenic surfaces with high or low epitope density, respectively. At high epitope density, IgM had a higher functional affinity for the Ag compared with IgD. The order was reversed at low epitope density due to a decrease in the functional affinity of IgM. Studies of binding kinetics showed similar association rates for both molecules. The dissociation rate, however, was slower for IgM at high epitope density and for IgD at low epitope density. Taken together, the results show that IgM and IgD with identical Ag binding regions have different Ag binding properties.  (+info)

The influence of immune complex-bearing follicular dendritic cells on the IgM response, Ig class switching, and production of high affinity IgG. (8/23)

It is believed that Ag in immune complexes (ICs) on follicular dendritic cells (FDCs) selects high affinity B cells and promotes affinity maturation. However, selection has been documented in the absence of readily detectable ICs on FDCs, suggesting that FDC-ICs may not be important. These results prompted experiments to test the hypothesis that IC-bearing murine FDCs can promote high affinity IgG responses by selecting B cells after stimulating naive IgM(+) cells to mature and class switch. Coculturing naive lambda(+) B cells, FDCs, (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (CGG) + anti-CGG ICs, and CGG-primed T cells resulted in FDC-lymphocyte clusters and production of anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl. Class switching was indicated by a shift from IgM to IgG, and affinity maturation was indicated by a change from mostly low affinity IgM and IgG in the first week to virtually all high affinity IgG anti-4-hydroxy-5-iodo-3-nitrophenyl acetyl in the second week. Class switching and affinity maturation were easily detectable in the presence of FDCs bearing appropriate ICs, but not in the absence of FDCs. Free Ag plus FDCs resulted in low affinity IgG, but affinity maturation was only apparent when FDCs bore ICs. Class switching is activation-induced cytidine deaminase (AID) dependent, and blocking FDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediated enhancement of AID production and the IgG response. In short, these data support the concept that ICs on FDCs can promote AID production, class switching, and maturation of naive IgM(+) B cells, and further suggest that the IC-bearing FDCs help select high affinity B cells that produce high affinity IgG.  (+info)

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