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R Dalchau

Publications and source records attributed to R Dalchau.

At least 19 recordsLinked to original sources
PubMed1996

A three-cell cluster hypothesis for noncognate T-B collaboration via direct T cell recognition of allogeneic dendritic cells.

In this article, we propose that T cell help for B cells can occur via an unusual three-cell cluster, with recipient CD4+ T helper cells interacting via direct allorecognition with donor dendritic cell class II MHC antigens, recipient B cells interacting with MHC class I (or any other) antigen on the donor dendritic cell surface, and noncognate (i.e., antigen nonspecific) T-B collaboration. In this noncognate pathway, antigen processing by B cells is not required and T cell help is potent because of the high precursor T cell frequency for direct recognition of allogeneic class II MHC molecules. The data supporting this hypothesis are: 1. LEW rat strain recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts were shown to have no detectable antibody to donor class I MHC antigens at day 7 after grafting. By contrast, LEW recipients of normal (DAxLEW)F1 kidneys had strong antibody responses. 2. Consistent wih important role for donor dendritic cells in the early antibody response to donor class I MHC antigens was the finding that it was dependent on donor class II MHC antigens. PVG recipients, previously immunized with pure DA RT1.B class II MHC antigens, had virtually no antibody response to the class I MHC antigens of DA kidney allografts. 3. We confirmed the low and high responder status of PVG and LEW rats, respectively, to DA class I antigens by studying antibody responses to pure DA class I antigens. However, PVG and LEW recipients of DA kidney allografts did not differ in their antibody response to the donor DA class I MHC antigens. This is consistent with this response not requiring the processing and presentation of DA class I antigen by PVG recipients. 4. LEW recipients of interstitial dendritic cell-free (DAxLEW)F1 kidney allografts did eventually develop a strong antibody response to DA class I antigens, but this was delayed by several weeks. That this delayed antibody response was probably mediated by conventional T-B collaboration and that T help was rate limiting in this situation, was demonstrated by immunizing LEW recipients with a DA class I peptide. This markedly accelerated the kinetics of the antibody response to the dendritic cell-free (DAxLEW)F1 kidneys.

Amino Acid Sequence↗
PubMed1994

T-cell recognition of an allogeneic RT1-Dbu class II MHC peptide.

The allo-antibody response of several rat strains to an unconjugated synthetic 20 amino acid peptide derived from the alpha helical region of the RT1-Du beta chain was tested. The LEW (RT1l) and WAG (RT1u) strains produced little or no antibody; the PVG (RT1c) and DA (RT1av1) strains produced moderate amounts of antibody; while the BN (RT1n) strain produced strong primary and secondary antibody responses. This suggested that the BN strain was able to process and present the RT1-Dbu peptide on its class II molecules. In vitro proliferation studies demonstrated that LEW T cells did not respond to the peptide, whereas BN T cells responded strongly, and that the response in the BN strain was found only in the CD4+ T-cell subset. However, immunisation of BN rats with the RT1-Dbu peptide failed to cause any acceleration of rejection of WAG skin or kidney grafts. Moreover, BN rats primed with WAG skin and kidney grafts did not produce T cells reactive to the RT1-Dbu synthetic peptide. This suggests that the T-cell response of the BN strain to the synthetic major histocompatibility complex peptide was not relevant to the indirect T-cell allo-recognition response to naturally processed RT1-Du beta chains.

Amino Acid Sequence↗
PubMed1993

Indirect T cell allorecognition: a cyclosporin A resistant pathway for T cell help for antibody production to donor MHC antigens.

LEW (RT1(1)) rats were primed for indirect allorecognition of DA (RT1avl) MHC molecules by immunizing either with synthetic peptides corresponding to the polymorphic regions of the RT1.Aavl classical class I MHC molecule, or with the isolated, denatured chains of the RT1.A class I, RT1.B alpha class II and RT1.B beta class II MHC molecules of the DA strain. These primed LEW rats received DA kidney allografts and were treated after grafting with cyclosporin A. Unprimed LEW controls mount a vigorous rejection response to DA kidney allografts and produce a strong antibody response to DA class I MHC antigens. Both the rejection and the antibody responses are virtually completely suppressed by cyclosporin A treatment in these controls. Priming to indirect allorecognition of donor MHC antigens did not diminish the effectiveness of cyclosporin A in suppressing the acute rejection of DA kidney grafts, but cyclosporin A could not suppress the early antibody response to the grafts in the primed rats. This finding could be of importance in clinical transplantation, where antibody-mediated graft damage might play an important role in both acute vascular rejection and chronic rejection.

Animals↗
PubMed1993

Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily.

Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility. This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation patterns, but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species, the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed in the same tissue in different species is all the more striking, given the known variation between species in the amino acid sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates and that the Thy-1 polypeptide serves as an appropriate carrier for these structures.

Amino Acid Sequence↗
PubMed1992

Rejection of skin allografts by indirect allorecognition of donor class I major histocompatibility complex peptides.

LEW (RT1l) rats were immunized with peptides corresponding to the alpha helical region of the alpha 1 domain (peptide 1), the beta sheet of the alpha 2 domain (peptide 2), and the alpha helical region of the alpha 2 domain (peptide 3) of the RT1-Aav1 classical class I molecule of the DA (RT1av1) strain. The immunizations were without carriers, and the objective was to prime to indirect allorecognition without influencing direct recognition of the RT1-Aav1 molecule. The LEW rats mounted strong primary and secondary antibody responses to peptides 1 and 3, but only weak secondary responses to peptide 2. None of the antipeptide antibodies crossreacted with intact RT1-Aav1 class I molecules. The immunization also resulted in LEW antigen-presenting cell-dependent, CD4+ T cell proliferative responses, which were very strong against peptide 1 and weakest against peptide 2. LEW rats immunized with peptides 1 or 3, but most effectively with both peptides 1 and 3 together, showed accelerated rejection of DA skin allografts. This effect was not observed in LEW rats immunized with peptide 2. In response to the DA skin allograft, the peptide-immunized LEW rats showed markedly accelerated kinetics of antibody production to the intact RT1-Aav1 molecule. These data demonstrate that indirect allorecognition can play an important role in allograft rejection and have important implications for understanding allograft rejection and its regulation.

Amino Acid Sequence↗
PubMed1992

Allorecognition of isolated, denatured chains of class I and class II major histocompatibility complex molecules. Evidence for an important role for indirect allorecognition in transplantation.

Classical RT1-A class I and RT1-B class II major histocompatibility complex (MHC) molecules were purified from DA (RT1avl) spleens, and the individual chains separated and purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. LEW (RT1l) rats were immunized with the pure class I heavy chain, the RT1-B alpha chain and the RT1-B beta chain with the aim of priming to indirect allorecognition (i.e. after processing and presentation of DA MHC chains on LEW antigen-presenting cells) in the absence of any priming to direct allorecognition (i.e. to whole, undenatured, dimeric DA MHC molecules). LEW rats immunized with each of the three DA MHC chains produced alloantibodies to these chains, suggesting that indirect allorecognition did occur, because of the requirement for cognate recognition of B cells by T helper cells. This also demonstrated polymorphism of all three chains between the DA and LEW strains. The antibodies to the isolated, denatured MHC chains did not react to the whole MHC molecules on DA cells, with the possible exception of very weak reactions in some class I heavy chain-immunized rats. DA skin grafts placed on LEW recipients immunized with each of the DA MHC chains were rejected in an accelerated fashion. Following DA skin grafting, there was an accelerated production of antibodies to whole, undenatured class I MHC molecules, even in the LEW rats preimmunized with RT1-B alpha and RT1-B beta chains. These data suggest that indirect allorecognition can play an important role in the effector mechanisms of allograft rejection, and demonstrate T helper priming as one possible mechanism whereby this might be effective.

Animals↗
PubMed1992

T cell recognition of donor major histocompatibility complex class I peptides during allograft rejection.

LEW (RTI1) recipients of DA (RTIav1) skin and kidney allografts were tested for the capacity of their T lymphocytes to proliferate to three 22-24-amino acid peptides from the hypervariable regions of the RTI-Aav1 classical MHC class I molecule. Ten days after rejecting second-set DA kidney allografts, spleen cells (but interestingly not lymph node cells) from LEW recipients showed strong, LEW antigen-presenting cell-dependent, CD4+ T cell proliferation to peptide 1 (from the alpha helical region of the alpha 1 domain). CD8+ T cells showed no response to peptide 1. There was no response by the spleen cells to peptide 2 (from the beta sheet of the alpha 2 domain) or peptide 3 (from the alpha helical region of the alpha 2 domain). Immunization of LEW rats with pure RTI-Aav1 class I H chain in Freund's adjuvant gave responses identical to that seen after grafting, i.e. good CD4+ T cell proliferation to peptide 1, but none to peptides 2 and 3. However, immunization of LEW rats with peptides 1, 2 and 3 in Freund's adjuvant resulted in good CD4+ T cell proliferation responses to each of the peptides. These data demonstrate that indirect allorecognition can be stimulated by allograft rejection, and emphasize that the physiological processing of donor antigens will influence which peptides will be important in indirect allorecognition in transplantation.

Amino Acid Sequence↗
PubMed1992

Stimulation of CD4+ T lymphocytes by allogeneic MHC peptides presented on autologous antigen-presenting cells. Evidence of the indirect pathway of allorecognition in some strain combinations.

A preliminary analysis of the alloantibody response to free, unconjugated class I and class II MHC peptides in several rat and mouse strains was performed, to screen for an effective interaction between the allogeneic MHC peptides and recipient MHC molecules. The PVG rat strain was noted to produce very strong, MHC-restricted, primary and secondary responses to a synthetic peptide derived from the alpha helical region of the alpha 2 domain of an RT1.C/E class I MHC molecule of the DA strain. In vitro proliferation studies demonstrated that CD4+ but not CD8+ T cells of the PVG strain responded in a recipient APC-dependent manner to the peptide, whereas the BN strain (which showed no antibody response to this peptide) gave no T cell proliferation. Immunization of PVG rats with the peptide did not influence the rejection of DA skin allografts. The relevance of these studies to the possible mechanisms of allograft rejection by an indirect pathway are discussed.

Amino Acid Sequence↗
PubMed1989

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses.

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.

Animals↗
PubMed1989

Chemical composition and tissue distribution of the human CDw44 glycoprotein.

The CDw44 glycoprotein was purified from 2.3 x 10(11) CD3+ CD4+ CD8- T-chronic lymphocytic leukaemia (CLL) cells using F10-44-2 monoclonal antibody affinity chromatography, DEAE-Sepharose anion-exchange chromatography, passage down carboxymethyl (CM)-Sepharose cation-exchange columns, wheat germ lectin affinity chromatography and gel-permeation chromatography. On elution in non-ionic detergents from the DEAE column, two distinct peaks of antigen activity were obtained. The CDw44 glycoprotein in each peak was a glycoprotein of 85,000 MW, but the amino acid composition of the peaks was noticeably different. Carbohydrate compositions showed that each peak contained approximately 30% (w/w) carbohydrate, the composition suggesting both O-linked and complex N-linked glycans. Modulation studies with the F10-44-2 antibody on normal peripheral blood mononuclear cells (PBMC) demonstrated that the CDw44 glycoprotein of T cells consisted of one fraction that was readily modulated, and the other which was resistant to modulation. Detailed tissue distribution studies for CDw44 were performed using the F10-44-2 antibody on frozen sections of human tissues. CDw44 has a restricted tissue distribution, but is found on many highly diverse cell types (e.g. T lymphocytes, smooth muscle cells, some secretory glands, skin epithelial cells).

Amino Acid Sequence↗
PubMed1988

The Ina and Inb blood group antigens are located on a glycoprotein of 80,000 MW (the CDw44 glycoprotein) whose expression is influenced by the In(Lu) gene.

The Ina and Inb blood group antigens were found to be located on an erythrocyte membrane glycoprotein of 80,000 MW by immunoblotting with human anti-Ina and anti-Inb antibodies under non-reducing conditions. This glycoprotein is shown here to be identical to that defined by monoclonal antibodies to CDw44, and a new murine monoclonal antibody (BRIC 35) is added to this cluster. Experiments with endo-beta-galactosidase and Endo F preparations suggest that the glycoprotein contains one or more N-glycans but that these oligosaccharides do not contain extensive poly-N-acetyllactosaminyl sequences. Experiments using membranes prepared from sialidase-treated normal erythrocytes, from Tn erythrocytes and from Cad erythrocytes suggest that the glycoprotein does not contain a substantial content of O-glycans. The Inb antigen and the epitope defined by a murine monoclonal antibody (BRIC 35) show reduced expression on Lu(a-b-) erythrocytes which result from the effect of the dominant inhibitor gene In(Lu). Evidence is presented here that the Inb antigen is expressed on normal granulocytes and lymphocytes and on the haemopoietic cell lines HEL, K562 and HL-60, a lymphoblastoid cell line and lymphocytes from two patients with B-CLL.

Antibodies, Monoclonal↗
PubMed1986

Structural implications of the location and stability to proteolytic enzymes of immunodominant determinants of the human leukocyte common molecule.

In this report, we use 8 different mouse monoclonal antibodies to define 3 immunodominant determinants of the human leukocyte common (LC) antigen, and by defining the relative location and the stability to proteolytic enzymes of these determinants we make some structural predictions about the LC family of molecules. One lineage-restricted determinant is expressed predominantly on B lymphocytes and is located on the two higher (210 and 195) kDa bands of LC, with possibly some expression on the 180-kDa band. The two other determinants, one of which is a complex of 3 partially overlapping sites, are expressed on all leukocytes and found on all four (210, 195, 180, 160 kDa) LC bands. Pronase and trypsin treatment of peripheral blood lymphocytes and of Daudi cells gave complete cleavage of the lineage-restricted determinant from the cells, but the two common determinants were, surprisingly, left intact on the cell surface. By contrast, pronase and trypsin treatment of pure, detergent-solubilized LC resulted in rapid degradation of the common determinants, while the restricted determinant was sensitive to pronase but not to trypsin. Purification of the LC molecule from pronase-treated Daudi cells yielded a 130-kDa lentil lectin-binding protein, while undegraded LC from Daudi cells has a molecular mass of 210 kDa. Our results demonstrate that both common and restricted determinants are at least partly protein in nature and that the restricted determinant is external to and therefore on the presumed amino terminal side of the common determinants. Moreover, because LC has been reported to have a large (approximately 100 kDa) intracellular component, our data suggest the presence of a relatively small (approximately 25 kDa) membrane proximal domain containing both of the common LC determinants and at least one N-linked oligosaccharide chain. This domain is potentially susceptible to proteolytic cleavage, but it is interesting that on the intact cell it is both protected from proteolytic degradation and unable to be cleaved from the cell surface.

Antibodies, Monoclonal↗
PubMed1985

The human leucocyte-common (LC) molecule: dissection of leukaemias using monoclonal antibodies directed against framework and restricted antigenic determinants.

Monoclonal antibodies have previously been raised against two separate antigenic determinants on the human LC molecule. One, F10.89.4, recognizes a 'framework' epitope on all LC molecules; these are found on the majority of leucocytes. The other, F8.11.13, recognizes only a 'restricted' epitope present on a subset of these molecules; this subset is found on B lymphocytes and a subpopulation of T lymphocytes. LC molecules on myeloid cells do not carry the 'restricted' antigenic determinant. We have investigated the differential expression of these LC epitopes on human leukaemias, using immunofluorescence on fresh leukaemic blasts and established cell lines. Our study shows that, as on normal haemopoietic cells, LC molecules on B leukaemias bear both 'framework' and 'restricted' epitopes, while the majority of T leukaemias bear only the 'framework' determinant. The small proportion of T cells that are F8.11.13+ ('restricted' epitope) are relatively mature, being of either OKT4+ or OKT8+ phenotype, and may be in an activated state (HLA-DR+). However, in contrast to normal haemopoietic cells, some myeloid leukaemias carry both 'framework' and 'restricted' epitopes (30% AML and AMML samples are F10.89.4+, F8.11.13+), and it is within this group that all TdT+ AML and AMML cases lie. Thus, these monoclonal antibodies should be useful for studying haemopoiesis in man and for analyzing human haemopoietic malignancies.

Adolescent↗
PubMed1983

Evidence for the occurrence of O-glycosidically linked oligosaccharides of poly-N-acetyllactosamine type on the human leucocyte common antigen.

High molecular weight glycoproteins of human B and T lymphocytes known as leucocyte common antigen or T200 have been shown to carry O- and N-glycosidically linked, sialylated, carbohydrate chains. The O-linked chains are polydisperse and those of B rather than T cell type are highly susceptible to degradation by endo-beta-galactosidase. These differences among lymphocytes that are functionally distinct raise the possibility that the oligosaccharides may contribute to the functions of these differentiation molecules as well as to their electrophoretic diversity.

Antigens↗