Role of Qa-1(b)-binding receptors in the specificity of developing NK cells

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.     

Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed.     

Biased amino acid distributions in regions of the T cell receptors and MHC molecules potentially involved in their association

We have analysed, in the context of the available structural information, the frequency of occurrence of different amino acids in functional regions of both the class I MHC antigens and of the TCR alpha and beta chains. We found that in class I MHC molecules, charged residues are found frequently among those which are presumably dedicated to interactions with the TCR, while the aromatic side chain residues are found more in the interior of the groove. In the TCR, the Asn residue appears with high frequency in all the CDR equivalents. The TCR CDR3s of both alpha and beta chains are particularly rich in Gly, whereas the CDR1 and CDR2 loops exhibit strong biases in favour of charged residues. Accordingly, the interactions between the MHC molecule and the peptide antigen appear to be essentially mediated by hydrophobic interactions and hydrogen bonding, while electrostatic interactions between charged residues might be important in the association of TCR and MHC molecules. The observation that each CDR1 and CDR2 is biased towards a particular set of amino acids, taken together with the nature of the protruding residues on the MHC helices, allows us to propose, in the frame of a molecular model of the MHC-TCR complex, several plausible configurations.     

Maternal immunoglobulins have no effect on the rate of maturation of the B cell compartment of the offspring

To elucidate a general role of maternal immunoglobulins (Ig) on the kinetics of B cell development of the offspring, we studied non-genetic influences of maternal Ig on the developing immune system of B cell-competent mice. These animals were the offsprings of either B cell-deprived μMT or of normal C57BL/6 females. In these mice, we have compared the kinetics of Ig production, the numbers of B cell progenitors, the expression of surface markers specific of the B lineage and the progression of Ig variable gene expression. We show that the absence of maternal Ig has no detectable effect on the kinetics of IgM and IgG production by the offspring's immune system. The number of B cell precursors, the kinetics of generation of B cells and their pattern of surface markers expression is identical in both types of mice. The acquisition of diversity in the B cell repertoire and the changes in the ratios of variable gene family expression are also indistinguishable. We conclude that maternally derived Ig has no influence on the rate of development and maturation of the B cell compartment of the offspring.     

The Diversity of Antigen-Specific TCR Repertoires Reflects the Relative Complexity of Epitopes Recognized

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 K^d. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252–260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170–179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant H-2 K^d molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/K^d complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

Expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.

The murine beta 2-microglobulina cDNA was cloned into pAc373 and pVL941 transfer vectors and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. Both types of recombinant baculoviruses were isolated and used to infect Spodoptera frugiperda (Sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pVL941-derived virus than when the pAc373-based virus was used. beta 2m was secreted into the culture medium where it accumulated and, under the best conditions, reached an approximate level of 10 micrograms/10(6) cells. Pulse-chase experiments after metabolic labelling with 35S-methionine followed by immunoprecipitation showed that beta 2m was stable, but that the secretion process in infected cells was relatively slow. Recombinant beta 2m was endowed with biological activity and was indistinguishable from that produced by mouse cells in 2D gel analysis. beta 2m was purified to near homogeneity from serum-free culture medium conditioned by recombinant baculovirus-infected cells by using an immunoaffinity column. The use of the insect cell/baculovirus expression system should constitute a suitable source of mouse beta 2m and should aid experiments aimed at unraveling its interactions with mouse class I histocompatibility molecules.     

Nodular glomerulosclerosis with deposition of monoclonal immunoglobulin heavy chains lacking C(H)1

The objective of this study was to further characterize the clinical and immunopathologic features of heavy chain deposition disease (HCDD), a recently described entity. Four patients were diagnosed as having HCDD on a kidney biopsy. All presented with nodular glomerulosclerosis with deposition of gamma1 heavy chains lacking CH1 epitopes, but without light chains. Two different patterns were observed in the serum. First, patients 1 and 2 had a circulating monoclonal IgGlambda containing a short gamma1 heavy chain lacking CH1 epitopes, with an apparent molecular weight of 40 kD consistent with a complete CH1 deletion. Biosynthetic experiments also showed that the deleted heavy chain was produced in excess compared with light chains, and was secreted in vitro together with half Ig molecules, although these abnormal components were not detected by Western blot analysis of whole serum. Second, patients 3 and 4 had a circulating monoclonal IgG1lambda with an apparently normal, nondeleted heavy chain subunit, but serum fractionation followed by immunoblotting revealed an isolated monoclonal gamma1 chain lacking CH1 epitopes. These data strongly suggest that renal deposition of a CH1-deleted heavy chain circulating in low amounts in the serum as a free unassembled subunit is a major feature of HCDD. The CH1 deletion is most likely responsible for the premature secretion in blood of the heavy chain by a clone of plasma cells.     

A prospective study on the evolution of the T-cell repertoire in patients with Sézary syndrome treated by extracorporeal photopheresis

Sézary syndrome is a leukemic form of epidermotropic cutaneous T-cell lymphoma related to the malignant proliferation of clonal CD4(+) T cells. Extracorporeal photochemotherapy may induce a transient improvement of the clinical signs, but its efficiency is discussed. To investigate the frequency of the T-cell clone in the peripheral blood of patients with Sézary syndrome and to monitor its evolution in patients treated using extracorporeal photopheresis or chemotherapy, we used the immunoscope technique. In one patient, we observed a decrease of the relative frequency of the clone from 15.6% to 0%, paralleling a complete remission of the clinical disease and a disappearance of the circulating Sézary cells. In the other cases, the evolution of the relative frequency paralleled the initial improvement of the clinical status and the absence of long-term efficiency in patients treated with extracorporeal photopheresis or chemotherapy. We observed a quick-acting direct cytotoxicity of the association 8MOP + UVA on the T-cell clone. The immunoscope technique appears to be an efficient tool to appreciate the amount of tumoral cells and to monitor the evolution of the clonal component in the Sézary syndrome.