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S Sidhu

Publications and source records attributed to S Sidhu.

At least 55 records · Page 3Linked to original sources
PubMed1992

Human T cells cannot act as autonomous antigen-presenting cells, but induce tolerance in antigen-specific and alloreactive responder cells.

The ability of two HLA-DR-expressing human T cell clones to function as antigen-presenting cells (APC) was investigated using highly purified T cells. The results demonstrated that these T cell clones are unable to act as autonomous APC, and that recognition of nominal or alloantigens on the surface of T cells leads to a state of nonresponsiveness. The first observation was that a T cell clone with specificity for the 306-324 peptide of influenza hemagglutinin (HA), and raised from a DR1 responder, exhibited apparent degeneracy of major histocompatibility complex restriction when cultured with peptide in the presence of peripheral blood mononuclear cells (PBMC) expressing a wide variety of structurally unrelated DR types. However, when the PBMC were pulsed with peptide and washed before coculture with the clone, peptide was exclusively recognized with DR1Dw1. This implied that in the presence of soluble peptide the T cells were displaying ligand to each other, and that the third-party APC were providing costimulatory signals. To test the ability of T cells to act as autonomous APC, accessory cell-free preparations of two DR1-restricted clones were cultured with peptide in the presence or the absence of added B cell APC. T cell purity was established by the absence of proliferation in response to the mitogen phytohemagglutinin (PHA). PHA-nonresponsive T cells were completely unable to proliferate in response to peptide alone; furthermore, preculture of the HA-specific clone, in the complete absence of accessory cells, with the same concentration of peptide (1 microgram/ml) that induced optimal proliferation when presented by conventional APC, led to profound nonresponsiveness. The same phenomenon was also observed when two of three anti-DR1 alloreactive T cell clones were precultured with a DR1-expressing T cell clone. The ability of the DR1-expressing clone to induce nonresponsiveness in anti-DR1 clones correlated with recognition of the DR1 alloantigen on the DR1-expressing clone.

Antigen-Presenting Cells↗
PubMed1991

Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence.

A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.

Amino Acid Sequence↗
PubMed1991

Limited regions of the alpha 2-domain alpha-helix control anti-A2 allorecognition: an analysis using a panel of A2 mutants.

The regions of the HLA-A2 molecule controlling anti-A2 alloreactivity were explored using naturally occurring allelic variants of HLA-A, and a panel of transfectants expressing the products of A2.1 genes that had been mutated at multiple positions encoding residues in the alpha 2 domain alpha-helix. As a means of detecting distant conformational effects, these altered A2.1 molecules were also examined serologically. Amino acid substitutions at the carboxy-terminal end of the alpha 2 domain alpha-helix led to diminished staining with the monoclonal antibody (mAb) MA2.1. The epitope for this antibody has previously been mapped to the alpha 1 domain alpha-helix (residues 62-65). This suggests that interdomain contacts may cause conformational alteration, and that mutants can have distant, as well as local effects. Of the 24 positions where substitutions were made, only six led to loss of the anti-A2 alloresponse by the three clones and three lines that were tested. In addition, the mutations that altered the MA2.1 epitope, located on the alpha 1 domain alpha-helix, did not inhibit allorecognition. This suggests that a limited number of regions on the A2.1 molecule are responsible for allodeterminant expression. The most influential substitutions were those at positions 152, 154, 162, and 166. It is notable that three of these are predicted to be T-cell receptor (Tcr)-contacting residues, and one (152) to contribute to peptide binding. These results suggest that the specificity of alloreactive T cells is determined by exposed polymorphisms, directly contacted by the Tcr, and by concealed polymorphisms which influence peptide binding.

Cell Line↗
PubMed1991

A monoclonal antibody with broad anti-HLA-DR activity fails to bind to DRw11/Dw5: possible effect of a unique polymorphism on the beta 1 domain alpha-helix.

We describe the generation and characterization of a murine monoclonal antibody with broad anti-HLA-DR beta activity, but which does not recognize DRw11/Dw5. A BALB/c mouse was immunized with a human alloreactive T-cell clone, with the intention of generating monoclonal anti-T-cell-receptor antibodies. In the course of screening the resulting hybridomas, a clone was detected which secreted an antibody (MP2) with anti-HLA-DR activity. This was shown by flow cytometry as well as immunoprecipitation followed by gel electrophoresis. Flow cytometry experiments using transfectants bearing hybrid human/murine class II molecules demonstrated that MP2 binds to the DR beta chain. MP2 bound to a wide range of Epstein-Barr-Virus-transformed cell lines and transfectants expressing different DR beta 1 and DR beta 3 subtypes: the only exceptions were three transfectants expressing DRw11/Dw5. One of these (RGT1) was shown to be functionally normal in DRw11-restricted alloreactive and antigen-specific systems. The specificity of MP2 was confirmed in functional assays: it was able to inhibit the recognition of DR1 and DRw15 but not DRw11 by alloreactive T-cell clones. Previously reported sequence data show that the beta chain of DRw11 differs from all other DR and DQ beta chains at position 58 by a glutamic acid for alanine substitution. This may account for the inability of DRw11/Dw5 to be recognized by MP2. The data emphasize the value of transfectants in defining precisely the allelic and chain specificity of anti-major-histocompatibility-complex (MHC) antibodies, and illustrate the influence that inaccessible residues can have on the conformation of MHC molecules.

Antibodies, Monoclonal↗
PubMed1991

The specificity of alloreactive T cells is determined by MHC polymorphisms which contact the T cell receptor and which influence peptide binding.

The separate contributions to allorecognition of peptide-binding and T cell receptor-contacting residues of an allogeneic HLA-DR molecule were investigated by site-directed mutagenesis. Alloreactive T cell clones were generated from a combination of responder (DR1Dw1,DR4Dw14) and stimulator (DR1Dw1, DR4Dw10) whose DR products differed at only three amino acid positions, two of which are predicted to interact with the T cell receptor (67 and 70), and one with bound peptide (71). Transfected murine DAP.3 cells expressing the wild type and mutated forms of DR4Dw10 in which the codons for residues 70 and/or 71 had been altered towards DR4Dw14 were used to stimulate a panel of anti-DR4Dw10 T cell clones. Substitutions at either position 70 or 71, or the combination of the two, led to loss of recognition by the alloreactive T cell clones. This implies that residues involved in peptide binding and residues involved in interaction with the T cell receptor are important for this panel of alloreactive T cell clones. The specificity of these alloreactive T cells for exposed polymorphic residues on the allogeneic MHC molecule was further demonstrated by the inhibitory effects of synthetic peptides, derived from the alpha-helix of the beta 1 domain of the DR4Dw10 molecule.

Animals↗
PubMed1990

Are primary alloresponses truly primary?

Proliferative T cell responses against major histocompatibility complex (MHC) incompatible stimulator cells in the mixed lymphocyte reaction are conventionally regarded as primary. However, it is generally accepted that the recognition of allogeneic MHC products results from a cross-reaction by self-MHC-restricted cells. These two assumptions were tested by examining the contribution of previously primed and naive T cells to 'primary' alloresponses. Peripheral blood T cells were separated into LFA-3+, memory, and LFA-3-, naive, populations by fluorescence-activated cell sorting. In contrast, to recall antigen responses to Candida albicans which were almost entirely confined to the LFA-3+, memory, population, the proliferative response to MHC incompatible stimulator cells, including HLA-DR-expressing mouse L cell transfectants, was equally distributed between the two T cell subsets in 5 day assays. Furthermore, limiting dilution analysis showed that the frequency of alloreactive T cells did not differ significantly between the two populations. The kinetics of proliferation in the two populations differed but were consistent with their naive and memory phenotype, in that after 3 days of culture the LFA-3+ cells proliferated more strongly to MHC alloantigens. These results show that a substantial proportion of 'primary' alloresponses are contributed by previously primed cells. In addition, the evidence for the cross-reactive hypothesis is supported and extended from the clonal to the population level.

Animals↗
PubMed1989

Co-recognition of endogenous antigens with HLA-DR1 by alloreactive human T cell clones.

The fine specificity of anti-HLA-DR1 alloreactive, human T cells was investigated by using DR1-expressing human and murine stimulator cells. All three bulk cell lines and six out of seven T cell clones proliferated in response to DR1-expressing mouse L cells. In addition to these species non specific T cells, three clones were identified which proliferated only in response to DR1 expressed by human or by murine stimulator cells. The patterns of response of these clones may reflect specificity for species or lineage-specific peptides with DR1. The results of aldehyde fixation and cytotoxicity experiments suggested that some of the T cell clones which proliferated in response to human and murine DR1 stimulators also required to recognize species-specific antigens. The responses of four of the six clones were abolished by fixation of DR1-L cells but not of a DR-1 EBV transformed lymphoblastoid cell line before co-culture. In addition, these clones were also cytotoxic for DR1-expressing human targets. The same clones which failed to recognize fixed L cells also failed to lyse DR1-L cells in a short term chromium release assay. Taken together these results suggest that some alloreactive anti-DR1, T cells are specific for peptides of cellular proteins seen in the context of the allo-MHC molecule. It is envisaged that L cells when co-cultured with human T cells, process and present peptides derived from proteins that are shed or secreted by the human cells, for co-recognition with DR1 on the L cell surface. The presentation of multiple peptides derived from endogenous proteins by allogeneic cells may contribute to the high precursor frequency of allo-reactive T cells.

Animals↗
PubMed1989

Allorecognition of DR1 by T cells from a DR4/DRw13 responder mimics self-restricted recognition of endogenous peptides.

Coculture of a series of anti-DR1Dw1 alloreactive human T-cell clones with autologous (DR4Dw4/DRw13Dw19) antigen-presenting cells and a series of recall antigens revealed that two of four clones tested proliferated in response to Candida albicans. One was restricted by DR4Dw4 and the other was restricted by DRw13Dw19. These results provide further evidence that many alloreactive T cells have a primary self-restricted specificity and cross-react on allogeneic major histocompatibility complex products. Structural comparison of the responder and stimulator DR molecules for these clones revealed that the regions predicted to contact the T cells' receptor, and thereby to determine self-restriction, are identical in sequence for DR4Dw4 and DR1Dw1 and differ by one residue between DRw13Dw19 and DR1Dw1. The DR beta residues that differ between responder, DR4Dw4 and DR13Dw19, and stimulator, DR1Dw1, are predicted to contribute to antigen binding. This implies that these anti-DR1 T cells may be specific for endogenous peptides that are bound by DR1 and not by the responder DR products, seen in a self-restricted manner. These T-cell clones also cross-reacted on DR4Dw13 and DRw14Dw16 molecules and on a human/murine hybrid class II dimer DR1 beta/I-E alpha. These reactions are discussed in terms of self-restricted peptide recognition. Thus these data suggest that in certain responder/stimulator combinations allorecognition may resemble self-restricted recognition of fragments of endogenous antigens that are bound by stimulator but not by responder major histocompatibility complex products.

Amino Acid Sequence↗
PubMed1987

A two-site sandwich immunoradiometric assay of human lymphotoxin with monoclonal antibodies and its applications.

Three monoclonal antibodies (MoAbs L49-15, L81-11 and L238-14) were raised against recombinant human lymphotoxin (rLT) derived from E. coli containing the cDNA sequence specifying LT. MoAb L81-11 strongly neutralised the cytotoxicity of LT derived either from E. coli or the RPMI 1788 lymphoblastoid cell line, whilst the other two MoAbs were only weakly neutralising in this respect. L81-11 and L238-14 MoAbs bound to different antigenic determinants on the rLT molecule, but neither bound to other lymphokines such as the structurally related tumour necrosis factor (TNF). As such, these MoAbs were ideal reagents for immunoassay of LT and a very sensitive, highly specific immunoradiometric assay (IRMA) was developed. This assay was rapid to perform and was capable of detecting as little as 10 pg/ml of LT. Application of the LT IRMA in combination with previously developed human gamma-interferon (IFN-gamma) and human TNF-specific IRMA (Crane et al., 1985; Meager et al., 1987) permitted independent estimations of these three substances to be carried out in parallel. By these means, it was found that RPMI 1788 produced both LT and TNF, but not IFN-gamma. Extensive analyses on cytokine (monokine and lymphokine) preparations derived from a variety of activated lymphocytes are also reported. Co-production of LT, TNF and IFN-gamma was a common finding, even occurring in alloantigen-specific T helper cell clones.

Antibodies, Monoclonal↗
PubMedDate not supplied

Hazardous air pollutants formation from reactions of raw meal organics in cement kilns.

Thermally induced chlorination, condensation, and formation reactions of raw meal organic surrogates were investigated on different types of surfaces. The System for Thermal Diagnostic Studies provided a powerful tool to study these reactions under defined reaction conditions, which were related to typical conditions in the preheater zone of cement kiln. Experiments were conducted with benzene and benzene/myristic acid (C6H6/C13H27COOH) mixtures in a quartz reactor containing different chlorinating catalysts/reagents over a temperature range of 300-500 degrees C. Reaction products were trapped in-line and analyzed by GC-MS. A mixture of chlorides of calcium, potassium, aluminium and iron was highly effective for chlorination/condensation reactions of benzene and benzene/myristic acid mix at temperatures above 300 degrees C. The same behavior was observed only when calcium chloride and potassium chloride were used as chlorinating catalyst/reagent. This result showed that transition metal chlorides like FeCl3 are not necessary for chlorination/condensation of organics under post-combustion conditions. Methylene chloride was the major chlorinated product followed by chloroform and various other C1, C2 and C6 chlorinated products. Yields of chlorinated aliphatics were highest at 400 degrees C for both benzene and benzene/myristic acid mix. C6 products were mainly mono- to hexa-chlorinated benzenes with trace amounts of chlorinated phenols. The major chlorinated products observed in this study (i.e., methylene chloride, chloroform, chloroethanes and monochlorobenzene) were also present as major chlorinated hydrocarbons in the cement kiln field emission data.

Journal Article↗