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R Biassoni

Publications and source records attributed to R Biassoni.

At least 55 records · Page 3Linked to original sources
PubMed1997

Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor.

Natural killer (NK) cells express killer cell inhibitory receptors (KIRs) for major histocompatibility complex class I molecules. Engagement of these surface receptors inhibits NK cell cytotoxic programs. KIR can also be expressed on T cell subsets, and their engagement similarly results in inhibition of effector functions initiated by the CD3/T cell receptor complex. KIR genes belong to two distinct families: the immunoglobulin superfamily (IgSF KIRs) and dimeric C2 lectins (lectin-like KIRs). Whereas both IgSF (p58: CD158, p70, and p140) and lectin-like KIRs (CD94/NKG2A heterodimers) have been found in human, only lectin-like KIRs (all members of the Ly-49 family) have been described in the mouse. We have generated transgenic mice expressing an IgSF KIR, CD158b (p58.2), which recognizes HLA-Cw3. Our data show that CD158b is necessary and sufficient to confer specificity to NK cells, as well as to modulate T cell activation programs in vitro. In addition, we did not detect any adaptation of CD158b cell surface expression to that of HLA class I ligands in the CD158b x HLA-Cw3 double transgenic mice, in contrast to observations with Ly-49 in the mouse. Therefore, distinct strategies of selection/calibration appear to be used by IgSF and lectin-like KIRs. Finally, the transgenic expression of CD158b KIR prevents the in vivo rejection of H-2 mismatch bone marrow grafts, which express the cognate major histocompatibility class I HLA-Cw3 allele, demonstrating for the first time the in vivo implication of human IgSF KIRs in the negative regulation of NK cell function.

Animals↗
PubMed1997

Reconstituted killer cell inhibitory receptors for major histocompatibility complex class I molecules control mast cell activation induced via immunoreceptor tyrosine-based activation motifs.

Natural killer and T cells express at their surface, members of a multigenic family of killer cell inhibitory receptors (KIR) for major histocompatibility complex Class I molecules. KIR engagement leads to the inhibition of natural killer and T cell activation programs. We investigated here the functional reconstitution of KIR in a non-lymphoid cell type. Using stable transfection in the RBL-2H3 mast cell line, we demonstrated that (i) KIR can inhibit signals induced by FcepsilonRIgamma or CD3zeta polypeptides that bear immunoreceptor tyrosine-based activation motifs; (ii) two distinct immunoreceptor tyrosine-based inhibition motifs-bearing receptors, i.e. KIR and FcgammaRIIB, use distinct inhibitory pathways since KIR engagement inhibits the intracellular Ca2+ release from endoplasmic reticulum stores, in contrast to FcgammaRIIB, which only inhibits extracellular Ca2+ entry; (iii) KIR require co-ligation with an immunoreceptor tyrosine-based activation motif-dependent receptor to mediate their inhibitory function. This latter finding is central to the mechanism by which KIR selectively inhibit only the activatory receptors in close vicinity. Taken together our observations also contribute to define and extend the family of immunoreceptor tyrosine-based inhibition motif-bearing receptors involved in the negative control of cell activation.

Animals↗
PubMed1997

The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules.

CD94, a type II membrane protein containing a C-type lectin domain, has been shown to be involved in natural killer (NK) cell-mediated recognition of different HLA allotypes. The inhibitory form of the CD94 receptor has recently been identified by the specific monoclonal antibody (mAb) Z199. Herein, we demonstrate that the inhibitory receptor is in fact a complex formed by the covalent association of CD94 with the NKG2-A molecule (Mr approximately 43 kDa), another member of the C-type lectin superfamily, and that Z199 mAb specifically recognize NKG2-A molecules. Although the NKG2-A-encoding cDNA has been known for several years, the corresponding protein and its possible function remained undefined. Moreover, we show that the NKG2-B protein, an alternatively spliced product of the NKG2-A gene, can also assemble with CD94. Remarkably, both NKG2-A and NKG2-B proteins contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). This may provide the molecular basis of the inhibitory function mediated by the CD94/NKG2-A receptor complexes.

Antigens, CD↗
PubMed1997

Role of amino acid position 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules.

In an attempt to identify the amino acid position(s) of the HLA-C-specific p58.1/p50.1 natural killer cell receptors that determine the binding affinity for their ligand, we used soluble fusion proteins formed by the ectodomain of either receptor and the Fc portion of human IgG1. We show that the soluble p50.1 (activating) receptor binds weakly to 221-Cw4 transfectants. In contrast, the soluble p58.1 (inhibitory) receptor binds with high affinity. A single amino acid mutation at position 70, obtained by site-directed mutagenesis, was found to affect the binding affinity of both the p50.1 and the p58.1 receptors. Thus, substitution in p50.1 of lysine 70 by threonine (typical of the inhibitory p58.1 molecule) resulted in a dramatic increase in binding affinity, comparable to that of the p58.1 molecule. On the other hand, substitution of threonine 70 by lysine in p58.1 almost abolished binding to 221-Cw4 cells. Our present data indicate that a single amino acid difference greatly influences the p58.1/p50.1 affinity for their HLA-C ligand and suggests a possible role of position 70 as a contact site in the natural killer cell receptor/major histocompatibility complex class I interaction.

Amino Acid Sequence↗
PubMed1997

Reciprocal expression of CD70 and of its receptor, CD27, in human long term-activated T and natural killer (NK) cells: inverse regulation by cytokines and role in induction of cytotoxicity.

By transfection of COS cells with an expression vector containing CD70 cDNA we demonstrate that two previously described MoAbs (ED6 and LD6) recognize CD70. By means of these MoAbs, we show that the surface expression of CD70 inversely correlates with the expression of its receptor, CD27, on activated T and NK cell populations and clones, although a subpopulation of cells expressing low density of both molecules exists. In addition, culture in the presence of IL-4 significantly enhances CD27 and reduces CD70 surface expression in phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), while tumour necrosis factor-alpha (TNF-alpha) displays opposite effects, indicating that receptor and ligand are reciprocally regulated by these cytokines. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD27 and CD70 mRNA suggests a transcriptional control of CD27 antigen expression in T cell clones. In addition, we show by the use of a re-directed killing assay that in cytotoxic T cell receptor (TCR) alpha/beta+ T cell clones, CD27 molecule may be involved in the regulation of cytolytic functions and may act synergistically with CD2. Finally, CD70 also acts as a signal-transducing molecule in some activated CD70+ TCR gamma/delta+ T or NK cell clones. In conclusion, our data indicate that CD27 and CD70 molecules are differentially expressed and regulated on long term-activated T and NK cells and are involved in the control of cellular functions.

Antibodies, Monoclonal↗
PubMed1997

HLA-class I-specific inhibitory receptors in human cytolytic T lymphocytes: molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells.

A subset of cytolytic T lymphocytes has been shown to express receptors of the NK type (NKR) which can inhibit T cell cytotoxicity induced via the TCR-CD3 pathway. In this study, by the analysis of full length cDNA amplified from representative T cell clones, we show that NKR belonging either to the lg superfamily, including p58.1, p58.2, p70 and p140, or to the C-type lectin superfamily (CD94/NKG2A), display sequences which are identical to those of the corresponding NKR expressed by CD3-NK cells. Moreover, a fragment of cDNA encoding the NKG2A protein was consistently amplified from all CD94+ T cell clones analyzed. Since different NKR types can be expressed by T cells, we analyzed whether individual T cells could co-express more than one NKR. Analysis of either resting or activated (and cultured) T cell populations revealed that two or more NKR can be co-expressed by single T cells. Moreover, by the analysis of T cell clones, we show that co-expressed receptors are functional and can inhibit independently the TCR-induced cytolytic function. Finally, we investigated whether NKR+ T lymphocytes were also present in lymphoid tissues. No such cells were found in thymus or cord blood, thus further supporting the notion that they represent memory T cells. On the other hand, they were present in all the peripheral tissues analyzed including spleen, lymph nodes and tonsils.

Animals↗
PubMed1997

Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes.

Human NK cells express several specialized inhibitory receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Receptors specific for groups of HLA-C (p58), HLA-B (p70) and HLA-A (p140) alleles belong to the Ig superfamily with two or three Ig-like domains in their extracellular portion, and a long cytoplasmic tail containing ITIM motifs and associated with a non-polar transmembrane portion. In contrast, the CD94/ NKG2-A receptor complex is composed of type II proteins with a C-type lectin domain which displays a more broad specificity for different class I alleles. Recently, activatory forms of the HLA-C-specific receptors have been identified in some donors. They are virtually identical to the inhibitory forms in their extracellular portions, but display a short cytoplasmic tail lacking ITIM motifs associated with a Lys-containing transmembrane portion (p50). A subset of activated T-lymphocytes, primarily CD8+ and oligoclonal or monoclonal in nature, express NK-type class I-specific receptors. These receptors exert an inhibitory activity on T-cell receptor-mediated functions and may provide an important mechanism of down-regulation of T-cell responses.

HLA Antigens↗
PubMed1996

The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer.

Human natural killer (NK) cells express inhibitory receptors that are specific for different groups of HLA-C or HLA-B alleles. The majority of these receptors belong to the immunoglobulin (Ig) superfamily and are characterized by two or three extracellular Ig-like domains. Here we describe a novel inhibitory NK receptor that is specific for a group of HLA-A alleles. The HLA-A3-specific NK cell clone DP7 has been used for mice immunization. Two mAbs, termed Q66 and Q241, bound to the immunizing clone and stained only a subset of NK cell populations or clones. Among Q66 mAb-reactive clones, we further selected those that did not express any of the previously identified HLA-class I-specific NK receptors. These clones did not lyse HLA-A3+ (or -A11+) target cells, but lysis of these targets could be detected in the presence of Q66 or Q241 mAbs. On the other hand, target cells expressing other HLA-A alleles, including -A1, -A2, and -A24, were efficiently lysed. Moreover, none of the HLA-C or HLA-B alleles that were tested exerted a protective effect. Q66+, but not Q66- NK cell clones, expressed messenger RNA coding for a novel 3 Ig domain protein homologous to the HLA-C (p58) and HLA-B (p70) receptors. The corresponding cDNA (cl.1.1) was used to generate transient and stable transfectants in COS7 and NIH3T3 cell lines, respectively. Both types of transfectants were specifically stained by Q66 and Q241 mAbs. Since the cytoplasmic tail of Q66-reactive molecules was at least 11 amino acid longer than the other known p58/p70 molecules, we could generate an antiserum specific for the COOH-terminus of Q66-reactive molecules, termed PGP-3. PGP-3 immunoprecipitated, only from Q66+ NK cells, molecules displaying a molecular mass of 140 kD, under nonreducing conditions, which resolved, under reducing conditions, in a 70-kD band. Thus, differently from the other p58/p70 receptors, Q66-reactive molecules appear to be expressed as disulphide-linked dimers and were thus termed p140. The comparative analysis of the amino acid sequences of p58, p70, and p140 molecules revealed the existence of two cysteins proximal to the transmembrane region, only in the amino acid sequence of p140 molecules.

3T3 Cells↗
PubMed1996

Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases.

NK cells express cell surface receptors for MHC class I proteins (KIR). Engagement of these receptors inhibits NK cell cytotoxic programs. KIR can be expressed on T cells, and their engagement also results in inhibition of effector functions initiated by the CD3/TCR complex. While human KIR genes belong to the Ig gene superfamily, mouse KIR belong to a family of dimeric lectins. Despite these distinct evolutionary origins, we show here that both HLA-Cw3-specific human p58.183 receptors and H-2D d/k-specific mouse Ly49A receptors recruit the same protein tyrosine phosphatases, PTP1C and PTP1D, upon phosphorylation of critical intracytoplasmic tyrosine residues. These results document a common pathway by which diverse KIR can down-regulate NK and T cell activation programs, and further define the sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM), initially described in FcgammaRIIB1, and expressed in both human and mouse KIR.

Amino Acid Sequence↗
PubMed1996

Identification of a novel interleukin-15 (IL-15) transcript isoform generated by alternative splicing in human small cell lung cancer cell lines.

IL-15 is a cytokine promoting growth and differentiation of T, B and NK lymphocytes. By RT-PCR analysis, using primers allowing amplification of the entire IL-15 mRNA coding region, 9/11 small cell lung cancer (SCLC) cell lines displayed detectable IL-15 gene expression. In addition to the expected band sizing 524 bp, a larger band was also observed. Cloning and sequence analysis of the larger cDNA from two SCLC cell lines revealed a size of 643 hp due to the presence of additional 119 hp within the previously reported IL-15 cDNA sequence. The 119 hp sequence matched with an IL-15 genomic sequence downstream the IL-15 second coding exon and may represent a previously unreported alternative exon (exon A). The SCLC-associated IL-15 mRNA isoform has a shorter open reading frame (ORF) due to stop codons in exon A, followed by a new AUG codon. The predicted IL-15 precursor protein displays a shorter signal peptide but shares the same aminoacidic composition of mature IL-15 protein. A possible functional role of IL-15, different from 'IL-2-like' activity, in human tumours, is suggested.

Alternative Splicing↗
PubMed1996

The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions.

Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.

Amino Acid Sequence↗
PubMed1996

Expression of human NKRP1A by CD34+ immature thymocytes: NKRP1A-mediated regulation of proliferation and cytolytic activity.

In this study, we show that NKRP1A is expressed and functions on a subset of immature human thymocytes. We took advantage of the monoclonal antibody (mAb) 191B8 that was obtained by immunizing mice with cultured human thymocytes characterized by an immature surface phenotype [CD2- CD3- CD4- CD8- stem cell factor receptor (SCFR)+] and expressing cytoplasmic CD3 epsilon chain. The 191B8 antibody homogeneously reacted with the immunizing population but not with most unfractionated thymocytes. It stained a minor population of resting immature thymocytes co-expressing CD34, SCFR, or both. Following culture of the CD34+ or CD34- fractions of CD2- CD3- CD4- CD8- purified immature thymocytes with recombinant interleukin-2 (rIL-2), the 191B8-defined antigen was expressed on virtually all cells even when 191B8+ cells were removed from the starting population. On the other hand, no 191B8+ cells were detected in fresh or cultured thymocytes expressing a more mature phenotype. Biochemical analysis of 191B8 mAb-reactive molecules revealed, under non-reducing conditions, two bands displaying apparent molecular masses of 80 and 44 kDa and a single band of 44 kDa under reducing conditions. Digestion with proteases indicated that the 80-kDa form represented a homodimeric form of two 44-kDa molecules, while deglycosylation with N-glycanase suggested the existence of four N-glycosylation sites. Transfection of COS7 or NIH3T3 cells with hNKRP1A cDNA showed that the 191B8 mAb recognized NKRP1A as shown by both immunofluorescence analysis and immunoprecipitation experiments. Functional studies showed that the 191B8/NKRP1A molecule mediated strong inhibition of the cytolytic activity of cultured CD2- CD3- immature thymocytes against a panel of tumor target cells. More importantly, 191B8 mAb induced proliferation of CD2- CD3- fresh thymocytes which was not increased by rIL-2. Thus, we propose that NKRP1A molecules, which are expressed in highly immature thymocytes, may play a regulatory role in their growth and function.

Antigens, CD34↗
PubMed1996

A novel surface molecule homologous to the p58/p50 family of receptors is selectively expressed on a subset of human natural killer cells and induces both triggering of cell functions and proliferation.

Human natural killer (NK) cells express inhibitory (p58) or activatory (p50) receptors for HLA-C alleles. Here, we describe a novel member of the p58/p50 family that is expressed by a subset of NK cells in about one third of donors. This molecule, termed p50.3, mediates NK cell triggering as revealed by the induction of intracellular free calcium mobilization, cytokine release and cytotoxicity. In addition, anti-p50.3 monoclonal antibody (mAb) induced a selective, strong proliferation of p50.3+ NK cells in peripheral blood lymphocytes. Although p50.3 molecules do not appear to display an obvious HLA class I specificity, they are usually coexpressed with known inhibitory receptors for HLA class I alleles, mAb-mediated cross-linking of these receptors leads to inhibition of the anti-p50.3 mAb-induced NK cell activation and proliferation. Surface p50.3 molecules are glycoproteins of approximately 55-58 kDa which, upon deglycosylation, display a relative molecular mass of 36 kDa, similar to that of deglycosylated (activatory) p50 receptors. Analysis of the two-dimensional peptide maps of the 50.3 molecules revealed a high homology with the other HLA-C-specific p58/p50 receptors. The use of a set of oligodeoxynucleotide primers, previously shown to amplify the activatory (p50) forms of HLA-C-specific receptors, consistently amplified in p50.3+ clones a cDNA sequence termed KKA3. This sequence belongs to the p58/p50 multigene family, that encodes for a transmembrane protein specifically stained by anti-p50.3 mAb in cell transfectants. Similar to p50 molecules, the KKA3-encoded molecules are characterized by two extracellular immunoglobulin-like domains, by the presence of a lysine in the transmembrane region and a short (39 amino acids) cytoplasmic tail which does not contain immune receptor tyrosine-based activation motifs (ITAM)-like sequences.

Amino Acid Sequence↗
PubMed1996

The molecular basis of natural killer (NK) cell recognition and function.

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail associated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.

Antigen Presentation↗
PubMed1996

Receptors for HLA class-I molecules in human natural killer cells.

Natural killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more class I alleles leads to NK-mediated target cell lysis. During NK cell development, the class I-specific receptors have adapted to self-class I molecules on which they recognize epitopes shared by groups of class I alleles. As such, they may fail to recognize either self-molecules that bound unusual peptides or allogeneic class I molecules unrelated to self-alleles. Different types of receptors specific for groups of HLA-C or HLA-B alleles have been identified. While in most instances, they function as inhibiting receptors, an activating form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C- and HLA-B-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibiting form is characterized by a long cytoplasmic tail associated with a nonpolar transmembrane portion, the activating one has a short tail associated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49 that is a type II transmembrane protein characterized by a C type lectin domain. A subset of cytolytic T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and offer a valuable model to analyze the regulatory mechanisms involved in receptor-mediated cell activation and inactivation.

Animals↗
PubMed1995

Amino acid substitutions can influence the natural killer (NK)-mediated recognition of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by "group 2" or "group 1" NK clones.

Natural killer (NK) cells have been shown to express a clonally distributed ability to recognize HLA class I alleles. The previously defined NK clones belonging to "group 1" recognize HLA-C*0401 (Cw4) and other HLA-C alleles sharing Asn at position 77 and Lys at position 80. Conversely, the "group 2" NK clones recognize HLA-Cw*0302 (Cw3) and other HLA-C alleles characterized by Ser at position 77 and Asn at position 80. We assessed directly the involvement of these two residues in the capacity of NK cell clones to discriminate between the two groups of HLA-C alleles. To this end, Cw3 and Cw4 alleles were subjected to site-directed mutagenesis. Substitution of the amino acids typical of the Cw3 allele (Ser-77 and Asn-80) with those present in Cw4 (Asn-77 and Lys-80) resulted in a Cw3 mutant that was no longer recognized by group 2 NK cell clones, but that was recognized by group 1 clones. Analysis of Cw3 or Cw4 molecules containing single amino acid substitutions indicates roles for Lys-80 in recognition mediated by group 1 clones and for Ser-77 in recognition mediated by group 2 clones. These results demonstrate that NK-mediated specific recognition of HLA-C allotypes is affected by single natural amino acid substitutions at positions 77 and 80 of the heavy chain.

Amino Acid Sequence↗
PubMed1995

Receptors for HLA class I molecules in human NK cells.

Recent studies have shown that NK cells recognize HLA-class I molecules. Moreover, the analysis of NK cell clones has provided evidence that they are capable of discriminating between different groups of HLA alleles. HLA class I recognition generates a negative signal which inhibits the NK cell cytotoxicity, thus resulting in target cell protection. HLA-class I recognition is mediated by clonally distributed receptors, some of which have been identified, characterized and cloned. The first two identified receptors were shown to be specific for HLA-C alleles, each recognizing a group of alleles sharing two amino acidic positions (77 and 80) in the peptide binding groove. The HLA-C specific receptors are represented by two 58 Kd (p58) molecules that are highly homologous, as shown by both biochemical analysis and by the comparison of the corresponding genes. Two additional receptors have been recently identified, which recognize two distinct groups of HLA-B alleles. These receptors are represented by the CD94 and by the NKB1 molecules, recognizing the Bw6 and Bw4 supertypic specificities. Recent analysis of the surface receptors involved in NK cell triggering has provided evidence that class I specific NK receptors can, in some instances, induce NK cell triggering, thus contributing to the activatory pathway of NK cells.

HLA Antigens↗