Biomedical subjects

C Waltzinger

Publications and source records attributed to C Waltzinger.

28 records · Page 2Linked to original sources

PubMed1991

The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.

To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.

Alleles↗

PubMed1991

The majority of peripheral blood monoclonal IgM secreting cells are CD5 negative in three patients with mixed cryoglobulinemia.

The mixed cryoglobulinemia is considered to be a nonmalignant human B-cell proliferation that frequently produces a monoclonal IgM with anti-IgG activity (rheumatoid factor). Using murine monoclonal anti-idiotypic antibodies specific for private or minor idiotopes on monoclonal IgM from three patients suffering from nonmalignant mixed cryoglobulinemia, we investigated the presence of the CD5 antigen on the monoclonal IgM producing cells in these patients. It is shown by two-color cytofluorometric analysis that the majority of the peripheral blood monoclonal IgM rheumatoid factor secreting cells is CD5 negative in these three patients. One of the monoclonal rheumatoid factor K variable regions was sequenced at the protein level and belongs to the human VK III group, as a high proportion of monoclonal rheumatoid factors and some B-cell chronic lymphocytic leukemia (CLL) membrane bound Igs. Thus, despite the preferential use of similar VK genes and the absence of somatic mutation affecting these variable regions in both malignant B-cell CLL and nonmalignant mixed cryoglobulinemia, these proliferating B cells differ in the CD5 membrane expression.

Amino Acid Sequence↗

PubMed1990

Functional consequences of overexpressed Ia antigens in AK alpha/AK beta transgenic mice.

We report the creation and characterization of several transgenic mouse lines that carry genes coding for the Ak alpha or Ak beta MHC class II (or Ia) molecules. In all these lines, the transgenes are expressed at the RNA and protein level with correct tissue and cell type specificity. Crosses between certain of them yield progeny displaying very high surface levels of class II protein--roughly five times the normal amount--allowing us to evaluate the consequences of quantitative variation in Ia molecule density on the organization and function of the immune system. The effects appear rather limited: we detect subtle changes in thymic lymphocyte subpopulations, as well as an enhanced Ag presentation capacity in vitro. Yet, in vivo responses are largely unaffected, and Ia overexpression to such levels does not provoke lymphoproliferation, immunodeficiency, or autoimmunity.

Animals↗

PubMed1990

Neonatal mouse CD4+ mature thymocytes show responsiveness to interleukin 2 and interleukin 7: growth in vitro of negatively selected V beta 6- and V beta 11-expressing CD4+ cells from (C57BL/6 x DBA/2)F1 mice.

By three colour flow microfluorimetry, we have recently shown that neonatal mouse CD4 single positive thymocytes are a population of proliferating cells. Furthermore, analysis of CD4+ thymocytes from (C57BL/6 x DBA/2)F1 mice showed that they proliferate regardless of whether they express particular V beta-encoded TCR molecules (V beta 6 and V beta 11) that are undergoing intrathymic deletion. In this report, cell culture experiments demonstrate that unstimulated neonatal CD4+ thymocytes from such mice proliferate in vitro in response to a combination of r-IL-2 and r-IL-7. Simultaneous three colour analysis of V beta TCR, CD4 expression, and DNA content of these cultured cells shows that V beta 6+, -8+, and -11+ cells grow equally well. Experiments where cells were cultured overnight in unsupplemented medium did not reveal preferential loss of negatively selected (V beta 6+ and V beta 11+) subpopulations of CD4+ cells. Taken together, these results suggest that IL-2 and IL-7 play a role in the intrathymic proliferation of developing mature T cells.

Animals↗

PubMed1990

Studies on the immunological properties of oxysterols: in vivo actions of 7,25-dihydroxycholesterol upon murine peritoneal cells.

Oxygenated derivatives of cholesterol are potent immunosuppressors. It has been reported previously that 7,25-dihydroxycholesterol (7,25-OHC), synthesized in the URA31, strongly inhibits the early steps of T-cell activation. So far, the mechanisms underlying this type of effect have been mainly investigated in vitro, and the activity of these substances on the immune system has been poorly studied. This study describes that a single i.p. injection of 7,25-OHC induces a strong inflammatory response, consisting of a massive influx of macrophages and neutrophils into the abdominal cavity. Macrophages harvested from 7,25-OHC-treated mice express class II antigen to a lesser extent. Moreover, the 7,25-OHC treatment abolishes the class II induction by bacillus Calmette-Guérin (BCG) or interferon-gamma (IFN-gamma). The inflammatory process triggered by the oxysterol is not the consequence of a non-specific effect due, for instance, to the presence of crystals in the abdominal cavity. Moreover, treatments by inhibitors of the acid arachidonic cascade do not affect the peritoneal exudate cell (PEC) influx induced by these substances. This study could be an important contribution to the mechanism determining the oxysterol-induced immunosuppression.

Animals↗

PubMed1989

Restricted assembly of MHC class II molecules in transgenic mice.

Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype-mismatched A alpha:A beta complexes. These findings led to the speculation that pairing restrictions, if applied in a cell type-specific fashion, might be involved in various autoimmune phenomena. We have investigated pairing restrictions in vivo by analyzing transgenic mice that carry an Ak alpha chain, an Ak beta chain, or the Ak alpha:Ak beta complex on an H-2b or H-2s background. Our conclusion is that the assembly of haplotype-mismatched A alpha:A beta complexes is limited in vivo, and that this is equally true for all cell types examined, regardless of their role in the immune response.

Alleles↗

PubMed1989

In vitro modulation of leucocyte-function-associated antigen-1 expression on two leukemic cell lines.

Leukocyte-function-associated antigen-1 (LFA-1) expression on two widespread tumor cell lines: K562 (an erythroleukemia) and MOLT-4 (a T leukemia), was investigated using two monoclonal antibodies specific for the alpha chain of this surface antigen, and flow cytometry analysis. When K562 cells are in the exponential phase of growth, they display very low levels of LFA-1. By contrast, cells from the plateau phase exhibit a strong labelling, which disappears rapidly when they are allowed to resume division by changing the culture medium. Using the same experimental conditions, we failed to detect any LFA-1 expression on MOLT-4 cells. However, after stimulation of these cells by phorbol myristate acetate, we observed a significant labelling, which occurred within 2 days of treatment. The LFA-1 expression disappears progressively after removal of the phorbol ester. From these results it may be concluded that (a) LFA-1 expression can vary considerably according to the culture conditions, (b) the expression of this antigen on the surface of non-expressing variants can be induced by phorbol ester, and (c) in both cases, the change in expression can be reversed completely by replacing the culture medium or by removing phorbol myristate acetate from it.

Antigens, Differentiation↗

PubMed1988

Slot-machine mutagenesis of a polymorphic residue on the A kappa alpha-chain.

This study explores the limitations on variability at a polymorphic position of an MHC class II molecule. Using a convenient and rapid method termed "slot-machine mutagenesis," we have converted Glu75 on the A kappa-chain to 15 alternative amino acids. This residue is of interest because it is an immunodominant site on the A kappa alpha chain and because it participates in certain T cell epitopes. The wild-type and mutant A kappa alpha cDNA were transfected into L cells (together with the A kappa beta cDNA and a selection marker), and transfectants displaying high surface levels of the A kappa complex were selected and expanded. We sought to examine three questions: what is the effect of these mutations on the expression and overall conformation of the A alpha: A beta complex? How do these diverse mutations influence mAb epitopes for which Glu75 makes a direct contribution to specificity? Do such substitutions affect T cell recognition of the A kappa alpha:A kappa beta complex? The answers to these three questions are quite different. Position 75 of the A alpha chain can accommodate essentially all chemically divergent amino acids without major consequences for expression and overall A alpha:A beta structure. In contrast, mAb that recognize Glu75-dependent epitopes are extremely particular about the amino acid residing at this position. T cells are less fastidious: those that are affected by the mutations still recognize a number of substitutions. These data emphasize the tolerance of MHC molecules to evolutionary tampering.

Amino Acid Sequence↗

PubMed1988

The enhancer in an MHC class II gene, in vitro and in mouso.

The E alpha class II gene of the major histocompatibility complex is expressed in a variety of immunocompetent cells. Part of the control of tissue-specific expression is mediated by a block of sequences found far upstream of the transcriptional startsite; this stretch is necessary for expression in the B lymphocytes of transgenic mice, but largely dispensable elsewhere. We review the evidence for the role of this region in E alpha transcription in transgenic animals, as well as data from transfections into tissue-culture cells, which indicate that this region has non-specific enhancer activity. We discuss possible models to explain how a non-specific enhancer can participate in cell-specific control.

Animals↗

PubMed1986

Functional sites on Ia molecules: a molecular dissection of A alpha immunogenicity.

Ia antigens are polymorphic cell-surface molecules that control the immune response. We have begun to localize important functional sites on one of the Ia molecules, A alpha. Herein, we focus on the A alpha k and A alpha b alleles and ask what defines "b-ness" and "k-ness" for a panel of monoclonal antibodies. Two independent experimental strategies are employed: the ability of 12 monoclonal antibodies to recognize L cell transfectants bearing chimeric and mutant A alpha chains is assessed, and the amino acid sequences of A alpha chains expressed by immunoselected B lymphoma mutants are deduced. For each antibody, we identify a stretch of polymorphic residues critical for recognition; for several, we can pinpoint a single amino acid. Certain stretches of A alpha (depending on the allele) appear strikingly immunodominant.

Amino Acid Sequence↗